Methods of treating acquired hemophilia with anti-fibrinolytic loaded platelets

ABSTRACT

Provided herein are methods and compositions for treating acquired hemophilia with platelets and/or platelet derivatives. In some cases, the platelets and or platelet derivatives are loaded with an anti-fibrinolytic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/969,942, filed on Feb. 4, 2020, U.S. Provisional PatentApplication No. 62/980,850, filed on Feb. 24, 2020, and U.S. ProvisionalPatent Application No. 63/065,337, filed on Aug. 13, 2020, the contentsof which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

Provided herein are compositions and methods for use of platelets,platelet derivatives, or thrombosomes (e.g., freeze-dried plateletderivatives) as biological carriers of cargo, such as anti-fibrinolyticcompounds, also referred to herein as anti-fibrinolytic loadedplatelets, platelet derivatives, or thrombosomes.

BACKGROUND

Blood is a complex mixture of numerous components. In general, blood canbe described as comprising four main parts: red blood cells, white bloodcells, platelets, and plasma. The first three are cellular or cell-likecomponents, whereas the fourth (plasma) is a liquid component comprisinga wide and variable mixture of salts, proteins, and other factorsnecessary for numerous bodily functions. The components of blood can beseparated from each other by various methods. In general, differentialcentrifugation is most commonly used currently to separate the differentcomponents of blood based on size and, in some applications, density.

Unactivated platelets, which are also commonly referred to asthrombocytes, are small, often irregularly-shaped (e.g., discoidal orovoidal) megakaryocyte-derived components of blood that are involved inthe clotting process. They aid in protecting the body from excessiveblood loss due not only to trauma or injury, but to normal physiologicalactivity as well.

Platelets are considered crucial in normal hemostasis, providing thefirst line of defense against blood escaping from injured blood vessels.Platelets generally function by adhering to the lining of broken bloodvessels, in the process becoming activated, changing to an amorphousshape, and interacting with components of the clotting system that arepresent in plasma or are released by the platelets themselves or othercomponents of the blood. Purified platelets have found use in treatingsubjects with low platelet count (thrombocytopenia) and abnormalplatelet function (thrombasthenia). Concentrated platelets are oftenused to control bleeding after injury or during acquired plateletfunction defects or deficiencies, for example those occurring duringsurgery and those due to the presence of platelet inhibitors.

SUMMARY OF THE INVENTION

Platelet transfusion and anti-fibrinolytics are suitable for improvingtraumatic bleeding events (e.g., hemorrhage). Methods and compositionsprovided herein generally describe the use of platelets as biologicalcarriers of anti-fibrinolytic compounds, including but not limited to,ε-aminocaproic acid (EACA). Loading anti-fibrinolytics into plateletscan shield the anti-fibrinolytic from systemic exposure and metabolicprocesses as the platelet migrates to the site of injury. At the site ofinjury the drug can be released to enhance the stability of the formingclot. Anti-fibrinolytic (e.g., EACA) loaded platelets can becryopreserved for long term storage, can retain the internalized cargo(e.g., EACA) after thawing, and can release the anti-fibrinolytic inresponse to in vitro stimulation by endogenous platelet agonists.

Provided herein are methods and compositions that are suitable forimproving traumatic bleeding (e.g., hemorrhage) therapy by reducing thetherapeutic dose of anti-fibrinolytic and prevent unwanted side effectsas a result of off-site drug interactions. Also provided herein aremethods and compositions that are used to treat hemophilia, includingacquired hemophilia (e.g., hemophilia with an inhibitor antibody) withanti-fibrinolytic (e.g., EACA) loaded platelets. In another aspectmethods and compositions that can be used to treat hemophilia, includingacquired hemophilia (e.g., hemophilia with an inhibitor antibody) withthrombosomes (e.g., unloaded thrombosomes).

Provided herein are methods of treating a coagulopathy in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent. In some embodiments,the platelets or platelet derivatives can be loaded with ananti-fibrinolytic.

Provided herein are methods of treating a coagulopathy in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition. In some embodiments, the platelets or platelet derivativescan be loaded with an anti-fibrinolytic.

Provided herein are methods of restoring normal hemostasis in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent. In some embodiments,the platelets or platelet derivatives can be loaded with ananti-fibrinolytic.

Provided herein are methods of restoring normal hemostasis in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition. In some embodiments, the platelets or platelet derivativescan be loaded with an anti-fibrinolytic.

Provided herein are methods of preparing a subject for surgery, whereinthe subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent. In some embodiments,the platelets or platelet derivatives can be loaded with ananti-fibrinolytic.

Provided herein are methods of preparing a subject for surgery, whereinthe subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition. In some embodiments, the platelets or platelet derivativescan be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent. In some embodiments,the platelets or platelet derivatives can be loaded with ananti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition. In some embodiments, the platelets or platelet derivativescan be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemophilia in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent. In some embodiments,the platelets or platelet derivatives can be loaded with ananti-fibrinolytic.

Provided herein are methods of treating a hemophilia in a subject,wherein the subject is a subject need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition. In some embodiments, the platelets or platelet derivativescan be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject,including administering a therapeutically effective amount ofanti-fibrinolytic loaded platelets to the subject in need thereof.

Also provided herein are methods of treating acquired hemophilia A in asubject, wherein the subject is a subject need thereof, includingadministering a therapeutically effective amount of anti-fibrinolyticloaded platelets to the subject in need thereof.

Provided herein are methods of treating acquired hemophilia B in asubject, wherein the subject is a subject need thereof, includingadministering a therapeutically effective amount of anti-fibrinolyticloaded platelets to the subject in need thereof.

In some embodiments of any of the methods provided herein, theconcentration of the therapeutically effective amount ofanti-fibrinolytic loaded into the platelets is from about 100 μM toabout 10 mM.

Provided herein are methods of treating acquired hemophilia A in asubject, wherein the subject is a subject need thereof, includingadministering a therapeutically effective amount of unloadedthrombosomes to the subject in need thereof.

Also provided herein are methods of treating acquired hemophilia B in asubject, wherein the subject is a subject need thereof, includingadministering a therapeutically effective amount of unloadedthrombosomes to the subject in need thereof.

In some embodiments of administering a therapeutically effective amountof unloaded thrombosomes to a subject in need thereof, includes aconcentration of the therapeutically effective amount of unloadedthrombosomes from about 1×10² particles/kg to about 1×10¹³ particles/kg.

In another aspect provided herein are methods of preparinganti-fibrinolytic loaded platelets including contacting platelets withan anti-fibrinolytic and with a loading buffer including a salt, a base,a loading agent, and optionally at least one organic solvent, to formthe anti-fibrinolytic loaded platelets. In some embodiments (e.g., forunloaded platelets or platelet derivatives), a “loading buffer” may bealternatively called an “incubating agent”.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including providing platelets and contacting the plateletswith an anti-fibrinolytic and with a loading buffer including a salt, abase, a loading agent, and optionally at least one organic solvent, toform the anti-fibrinolytic loaded platelets. In some embodiments ofpreparing anti-fibrinolytic loaded platelets, the platelets arecontacted with the anti-fibrinolytic and with the loading buffersequentially, in either order.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including contacting platelets with the anti-fibrinolytic toform a first composition and contacting the first composition with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, to form the anti-fibrinolytic loadedplatelets.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including contacting the platelets with a buffer including asalt, a base, a loading agent, and optionally at least one organicsolvent to form a first composition and contacting the first compositionwith an anti-fibrinolytic, to form the anti-fibrinolytic loadedplatelets. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the platelets are contacted with the anti-fibrinolytic andwith the loading buffer concurrently.

In another aspect provided herein are methods of preparinganti-fibrinolytic loaded platelets, including contacting the plateletswith an anti-fibrinolytic in the presence of a loading buffer includinga salt, a base, a loading agent, and optionally at least one organicsolvent to form the anti-fibrinolytic loaded platelets. In someembodiments of preparing anti-fibrinolytic loaded, the platelets arepooled from a plurality of donors prior to a treating step.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including A) pooling platelets from a plurality of donors andB) contacting the platelets from step (A) with an anti-fibrinolytic andwith a loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, to form the anti-fibrinolyticloaded platelets.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including A) pooling platelets from a plurality of donors andB) contacting the platelets from step (A) with an anti-fibrinolytic toform a first composition and contacting the first composition with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, to form the anti-fibrinolytic loadedplatelets.

Provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including A) pooling platelets from a plurality of donors andB) contacting the platelets from step (A) with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form a first composition and contacting the firstcomposition with an anti-fibrinolytic to form the anti-fibrinolyticloaded platelets.

Also provided herein are methods of preparing anti-fibrinolytic loadedplatelets, including A) pooling platelets from a plurality of donors andB) contacting the platelets with an anti-fibrinolytic in the presence ofa loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, to form the anti-fibrinolyticloaded platelets. In some embodiments of preparing anti-fibrinolyticloaded platelets, the loading agent is a monosaccharide or adisaccharide. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the loading agent is sucrose, maltose, trehalose, glucose,mannose, or xylose. In some embodiments of preparing anti-fibrinolyticloaded platelets, the platelets are isolated prior to a contacting step.In some embodiments of preparing anti-fibrinolytic loaded platelets, theplatelets are loaded with the drug in a period of time of 5 minutes to48 hours. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the concentration of the anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 1 μM to about 100 mM.In some embodiments of preparing anti-fibrinolytic loaded platelets, theone or more organic solvents is selected from the group consisting ofethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran(THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinationsthereof. In some embodiments of preparing anti-fibrinolytic loadedplatelets, cold storing, cryopreserving, freeze-drying, thawing,rehydrating, and combinations thereof the anti-fibrinolytic loadedplatelets. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the drying step includes freeze-drying the anti-fibrinolyticloaded platelets. In some embodiments of preparing anti-fibrinolyticloaded platelets, further including rehydrating the anti-fibrinolyticloaded platelets obtained from the drying step.

Also provided herein are anti-fibrinolytic loaded platelets prepared byany of the methods described herein.

Also provided herein are rehydrated anti-fibrinolytic loaded plateletsprepared by a method including rehydrating the anti-fibrinolytic loadedplatelets by any of the methods described herein. In some embodiments ofpreparing anti-fibrinolytic loaded platelets, the anti-fibrinolytic ismodified with an imaging agent. In some embodiments of preparinganti-fibrinolytic loaded platelets, the anti-fibrinolytic is modifiedwith the imaging agent prior to contacting platelets with theanti-fibrinolytic. In some embodiments of preparing anti-fibrinolyticloaded platelets, the platelets are further treated with an imagingagent, where the anti-fibrinolytic loaded platelets are loaded with theimaging agent. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the method does not include contacting the platelets with anorganic solvent. In some embodiments of preparing anti-fibrinolyticloaded platelets, the method does not include contacting the firstcomposition with an organic solvent. In some embodiments of preparinganti-fibrinolytic loaded platelets, the anti-fibrinolytic is selectedfrom the group consisting of ε-aminocaproic acid, aprotinin,aminomethylbenzoic acid, tranexamic acid, and fibrinogen. In someembodiments of preparing anti-fibrinolytic loaded platelets, theε-aminocaproic acid is present in a concentration of at least 100 μM.

Provided herein are anti-fibrinolytic loaded platelets prepared by anyof the methods described herein.

Also provided herein are methods where the anti-fibrinolytic-loadedplatelets treat a hemorrhage in a subject, wherein the subject is asubject need thereof.

Also provided herein, are methods of preparing platelets, plateletderivatives, or thrombosomes loaded with anti-fibrinolytic compounds.Also provided herein, are methods of treating conditions, such ashemophilia, or conditions such as hemorrhaging with platelets, plateletderivatives, thrombosomes, and/or thrombosomes loaded withanti-fibrinolytic compounds. Also provided herein, are methods oftreating conditions such as acquired hemophilia, or conditions such ashemorrhaging (e.g., trauma) with unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes.

Also provided herein are methods and compositions that are suitable fortreating drug-induced coagulopathy, such as antiplatelet agent-inducedcoagulopathy, such as, for example, treatment with anti-fibrinolyticloaded platelets, anti-fibrinolytic loaded thrombosomes, oranti-fibrinolytic loaded platelet derivatives.

Also provided herein are methods and compositions that are suitable fortreating coagulopathy, such as a disease-caused coagulopathy (e.g.,acquired hemophilia) or a drug-induced coagulopathy, such as, forexample, treatment with anti-fibrinolytic loaded platelets,anti-fibrinolytic loaded thrombosomes, or anti-fibrinolytic loadedplatelet derivatives.

Also provided herein are methods and compositions that are suitable fortreating drug-induced coagulopathy, such as, for example, treatment withplatelets, thrombosomes, or platelet derivatives.

Also provided herein are methods and compositions that are suitable fortreating coagulopathy, such as antiplatelet agent-induced coagulopathy,such as for example, treatment with platelets, thrombosomes, or plateletderivatives.

Anti-fibrinolytic loaded platelets described herein can be stored undertypical ambient conditions, refrigerated, cryopreserved, for examplewith dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization(e.g., to form thrombosomes) Also provided herein are methods where theanti-fibrinolytic loaded platelets treat a disease in a subject, whereinthe subject is a subject need thereof. In some embodiments where theanti-fibrinolytic loaded platelets treat a disease, theanti-fibrinolytic loaded platelets treat hemophilia. In someembodiments, the hemophilia is acquired hemophilia.

Also provided herein are methods of treating a hemorrhage in a subject,wherein the subject is a subject need thereof, including administering atherapeutically effective amount of unloaded thrombosomes to the subjectin need thereof. In some embodiments of treating a hemorrhage in asubject, the concentration of the therapeutically effective amount ofunloaded thrombosomes is from about 1×10² particles/kg to about 1×10¹³particles/kg.

Also provided herein are methods of treating acquired Hemophilia A in asubject, wherein the subject is a subject need thereof, the methodincluding, administering a therapeutically effective amount of loadedthrombosomes to a subject in need thereof.

Also provided herein are methods of treating acquired Hemophilia B in asubject, wherein the subject is a subject need thereof, the methodincluding, administering a therapeutically effective amount of loadedthrombosomes to a subject in need thereof, wherein the loadedthrombosomes are loaded with an anti-fibrinolytic.

In some embodiments of any of the methods described herein, theanti-fibrinolytic is selected from the group including of ε-aminocaproicacid, aprotinin, aminomethylbenzoic acid, tranexamic acid, andfibrinogen.

In some embodiments, the anti-fibrinolytic is ε-aminocaproic acid. Insome embodiments, the ε-aminocaproic acid is present in a concentrationfrom about 1 μM to about 100 mM.

Also, provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding contacting platelets with an anti-fibrinolytic and with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, to form the composition, where the subjecthas been treated or is being treated with an anticoagulant.

Also, provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition includinganti-fibrinolytic loaded platelets, where the subject has been treatedor is being treated with an anticoagulant.

Also, provided herein are methods of restoring normal hemostasis in asubject, the method including administering to the subject in needthereof a therapeutically effective amount of a composition prepared bya process including contacting platelets with an anti-fibrinolytic andwith a loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, to form the composition, wherethe subject has been treated or is being treated with an anticoagulant.

Also, provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition including anti-fibrinolytic loadedplatelets, where the subject has been treated or is being treated withan anticoagulant.

Also, provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof an effectiveamount of a composition prepared by a process including contactingplatelets with an anti-fibrinolytic and with a loading buffer includinga salt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition, where the subject has been treated oris being treated with an anticoagulant.

In some embodiments of preparing a subject for surgery, the surgery isan emergency surgery. In some embodiments of preparing a subject forsurgery the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatmentwith the anticoagulant is stopped. In some embodiments of any of themethods described herein, treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof a therapeutically effective amount of anti-fibrinolyticloaded platelets.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof a therapeutically effective amount of a compositionprepared by a process including contacting platelets with ananti-fibrinolytic and with a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent, to form thecomposition.

In some embodiments of ameliorating the effects of anticoagulant in asubject, the composition is administered following administration to thesubject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, theanticoagulant is selected from the group consisting of dabigatran,argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux,warfarin, heparin, a low molecular weight heparin, a supplement, and acombination thereof.

In some embodiments of any of the methods described herein, theanticoagulant is selected from the group consisting of dabigatran,argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux,warfarin, heparin, low molecular weight heparins, tifacogin, FactorVIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889,idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban,lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol,indandiones, fluindione, and a supplement, and a combination thereof.

In some embodiments of any of the methods described herein, theanticoagulant is warfarin. In some embodiments of any of the methodsdescribed herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, the methodincludes drying the composition prior to the administration step. Insome embodiments of any of the methods described herein, the methodincludes rehydrating the composition following the drying step.

In some embodiments of any of the methods described herein, the methodincludes freeze-drying the composition prior to the administration step.In some embodiments of any of the methods described herein, the methodincludes rehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, one or moreorganic solvents is selected from the group consisting of ethanol,acetic acid, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran(THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinationsthereof. In some embodiments of any of the methods described herein, thecomposition includes an organic solvent.

In some embodiments of any of the methods described herein, theanti-fibrinolytic loaded platelets or anti-fibrinolytic loaded plateletderivatives includes thrombosomes.

Also provided here are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition includinganti-fibrinolytic loaded platelets or anti-fibrinolytic loaded plateletderivatives and a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, where the subjecthas been treated or is being treated with an antiplatelet agent.

Also provided here are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding contacting platelets with an anti-fibrinolytic and with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, to form the composition, where the subjecthas been treated or is being treated with an antiplatelet agent.

Also provided here are methods of treating a coagulopathy in a subject,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition including anti-fibrinolytic loadedplatelets or anti-fibrinolytic loaded platelet derivatives and a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, where the subject has been treated or isbeing treated with an antiplatelet agent.

Also provided here are methods of treating a coagulopathy in a subject,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition prepared by a process includingcontacting platelets with an anti-fibrinolytic and with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the composition, where the subject has beentreated or is being treated with an antiplatelet agent.

Also provided here are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition including anti-fibrinolytic loadedplatelets or anti-fibrinolytic loaded platelet derivatives and a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, where the subject has been treated or isbeing treated with an antiplatelet agent.

Also provided here are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition prepared by a process includingcontacting platelets with an anti-fibrinolytic and with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the composition, where the subject has beentreated or is being treated with an antiplatelet agent.

In some embodiments of preparing a subject for surgery, the surgery isan emergency surgery. In some embodiments of preparing a subject forsurgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatmentwith the antiplatelet agent is stopped. In some embodiments of any ofthe methods described herein, treatment with the antiplatelet agent iscontinued.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, including administering to the subjectin need thereof a therapeutically effective amount of a compositionincluding anti-fibrinolytic loaded platelets or anti-fibrinolytic loadedplatelet derivatives and a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, the method including administering tothe subject in need thereof a therapeutically effective amount of acomposition prepared by a process including contacting platelets with ananti-fibrinolytic and with a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent, to form thecomposition.

In some embodiments of ameliorating the effects of an antiplatelet agentin a subject, the composition is administered following administrationto the subject or assumption by subject, or an overdose of theantiplatelet agent.

In some embodiments of any of the methods described herein, theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, and a supplement, and a combination thereof. In someembodiments of any of the methods described herein, the antiplateletagent is selected from the group consisting of aspirin, cangrelor,ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab,terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar,and atopaxar, and a combination thereof. In some embodiments of any ofthe methods described herein, the antiplatelet agent is selected fromthe group consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol,prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, andsarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein, the methodincludes drying the composition prior to the administration step. Insome embodiments of any of the methods described herein, the methodincludes rehydrating the composition following the drying step. In someembodiments of any of the methods described herein, the method includesfreeze-drying the composition prior to the administration step. In someembodiments of any of the methods described herein, the method includesrehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, one or moreorganic solvents is selected from the group consisting of ethanol,acetic acid, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran(THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinationsthereof. In some embodiments of any of the methods described herein, thecomposition includes an organic solvent.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, where the subject has been treated or is being treated with ananticoagulant.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition, where the subject has beentreated or is being treated with an anticoagulant.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, where the subject has been treated or is being treated with ananticoagulant.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition, where the subject has beentreated or is being treated with an anticoagulant.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition including platelets or plateletderivatives and an incubating agent including one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent,where the subject has been treated or is being treated with ananticoagulant.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition prepared by a process includingincubating platelets with an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition, where the subject has been treated oris being treated with an anticoagulant.

In some embodiments of preparing a subject for surgery, the surgery isan emergency surgery. In some embodiments of preparing a subject forsurgery, the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, the subjector is being treated with an anticoagulant. In some embodiments of any ofthe methods described herein, treatment with the anticoagulant isstopped. In some embodiments of any of the methods described herein,treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof a therapeutically effective amount of a compositionincluding platelets or platelet derivatives and an incubating agentincluding one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof a therapeutically effective amount of a compositionprepared by a process including incubating platelets with an incubatingagent including one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition.

In some embodiments of ameliorating the effects of an anticoagulant in asubject, the composition is administered following administration to thesubject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, thecomposition includes an anti-fibrinolytic agent. In some embodiments ofany of the methods described herein, the anti-fibrinolytic agent isselected from the group consisting of ε-aminocaproic acid (EACA),tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and acombination thereof.

In some embodiments of any of the methods described herein, theanticoagulant is selected from the group consisting of dabigatran,argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux,warfarin, heparin, a low molecular weight heparin, and a supplement, anda combination thereof. In some embodiments of any of the methodsdescribed herein, the anticoagulant is selected from the groupconsisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban,edoxaban, fondaparinux, warfarin, heparin, low molecular weightheparins, tifacogin, Factor VIIai, SB249417, pegnivacogin (with orwithout anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A,apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran,phenprocoumon, acenocoumarol, indandiones, fluindione, and a supplement,and a combination thereof. In some embodiments of any of the methodsdescribed herein, the anticoagulant is warfarin. In some embodiments ofany of the methods described herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, beforeadministering, the subject had an INR of at least 4.0. In someembodiments of any of the methods described herein, after theadministering, the subject has an INR of 3.0 or less. In someembodiments of any of the methods described herein, after theadministering, the subject has an INR of 2.0 or less. In someembodiments of any of the methods described herein, beforeadministering, the subject had an INR of at least 3.0. In someembodiments of any of the methods described herein, after administering,the subject has an INR of 2.0 or less.

In some embodiments of any of the methods described herein,administering includes administering topically. In some embodiments ofany of the methods described herein, administering includesadministering parenterally. In some embodiments of any of the methodsdescribed herein, administering includes administering intravenously. Insome embodiments of any of the methods described herein, administeringincludes administering intramuscularly. In some embodiments of any ofthe methods described herein, administering includes administeringintrathecally. In some embodiments of any of the methods describedherein, administering includes administering subcutaneously. In someembodiments of any of the methods described herein, administeringincludes administering intraperitoneally.

In some embodiments of any of the methods described herein, the methodincludes drying the composition prior to the administration step. Insome embodiments of any of the methods described herein, the methodincludes rehydrating the composition is following the drying step. Insome embodiments of any of the methods described herein, the methodincludes freeze-drying the composition prior to the administration step.In some embodiments of any of the methods described herein, the methodincludes rehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, theincubating agent includes one or more salts selected from phosphatesalts, sodium salts, potassium salts, calcium salts, magnesium salts,and a combination of two or more thereof.

In some embodiments of any of the methods described herein, theincubating agent includes a carrier protein. In some embodiments of anyof the methods described herein, the buffer includes HEPES, sodiumbicarbonate (NaHCO₃), or a combination thereof. In some embodiments ofany of the methods described herein, the composition includes one ormore saccharides. In some embodiments of any of the methods describedherein, the one or more saccharides includes trehalose. In someembodiments of any of the methods described herein, the one or moresaccharides includes polysucrose. In some embodiments of any of themethods described herein, the one or more saccharides includes dextrose.

In some embodiments of any of the methods described herein, thecomposition includes an organic solvent.

In some embodiments of any of the methods described herein, theplatelets or platelet derivatives includes thrombosomes.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, where the subject has been treated or is being treated with anantiplatelet agent.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition, where the subject has beentreated or is being treated with an antiplatelet agent.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, where the subject has been treated or is being treated with anantiplatelet agent.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition, where the subject has beentreated or is being treated with an antiplatelet agent.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition including platelets or plateletderivatives and an incubating agent including one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent,where the subject has been treated or is being treated with anantiplatelet agent.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof a therapeuticallyeffective amount of a composition prepared by a process includingincubating platelets with an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition, where the subject has been treated oris being treated with an antiplatelet agent.

In some embodiments of preparing a subject for surgery, the surgery isan emergency surgery. In some embodiments of preparing a subject forsurgery, the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatmentwith the antiplatelet agent is stopped. In some embodiments of any ofthe methods described herein, treatment with the antiplatelet agent iscontinued.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, including administering to the subjectin need thereof a therapeutically effective amount of a compositionincluding platelets or platelet derivatives and an incubating agentincluding one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, including administering to the subjectin need thereof a therapeutically effective amount of a compositionprepared by a process including incubating platelets with an incubatingagent including one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition.

In some embodiments of ameliorating the effects of an antiplatelet agentin a subject, the composition is administered following administrationto the subject or assumption by subject, or an overdose of theantiplatelet agent.

In some embodiments of any of the methods described herein, thecomposition includes an anti-fibrinolytic agent. In some embodiments ofany of the methods described herein, the anti-fibrinolytic agent isselected from the group consisting of ε-aminocaproic acid (EACA),tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and acombination thereof.

In some embodiments of any of the methods described herein, theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, and a supplement, and a combination thereof. In someembodiments of any of the methods described herein, the antiplateletagent is selected from the group consisting of aspirin, cangrelor,ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab,terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar,and atopaxar, and a combination thereof. In some embodiments of any ofthe methods described herein, the antiplatelet agent is selected fromthe group consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol,prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, andsarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein,administering includes administering topically. In some embodiments ofany of the methods described herein, administering includesadministering parenterally. In some embodiments of any of the methodsdescribed herein, administering includes administering intravenously. Insome embodiments of any of the methods described herein, whereadministering includes administering intramuscularly. In someembodiments of any of the methods described herein, where administeringincludes administering intrathecally. In some embodiments of any of themethods described herein, where administering includes administeringsubcutaneously. In some embodiments of any of the methods describedherein, where administering includes administering intraperitoneally.

In some embodiments of any of the methods described herein, the methodincludes drying the composition prior to the administration step. Insome embodiments of any of the methods described herein, the methodincludes rehydrating the composition after the drying step. In someembodiments of any of the methods described herein, the method includesfreeze-drying the composition prior to the administration step. In someembodiments of any of the methods described herein, the methodrehydrating the composition is after the freeze-drying step.

In some embodiments of any of the methods described herein, theincubating agent includes one or more salts selected from phosphatesalts, sodium salts, potassium salts, calcium salts, magnesium salts,and a combination of two or more thereof. In some embodiments of any ofthe methods described herein, where the incubating agent includes acarrier protein. In some embodiments of any of the methods describedherein, the buffer includes HEPES, sodium bicarbonate (NaHCO₃), or acombination thereof.

In some embodiments of any of the methods described herein, where thecomposition includes one or more saccharides. In some embodiments of anyof the methods described herein, one or more saccharides includestrehalose. In some embodiments of any of the methods described herein,the one or more saccharides includes polysucrose. In some embodiments ofany of the methods described herein, the one or more saccharidesincludes dextrose.

In some embodiments of any of the methods described herein, thecomposition includes an organic solvent.

In some embodiments of any of the methods described herein, theplatelets or platelet derivatives includes thrombosomes.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof aneffective amount of a composition including platelets or plateletderivatives and an incubating agent including one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of treating a coagulopathy in asubject, including administering to the subject in need thereof aneffective amount of a composition prepared by a process includingincubating platelets with an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

In some embodiments of treating a coagulopathy in a subject, thecomposition is administered following administration to the subject anantiplatelet agent or an anticoagulant, or a subject having VonWillebrand Disease or hemophilia.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof aneffective amount of a composition including platelets or plateletderivatives and an incubating agent including one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of restoring normal hemostasis in asubject, including administering to the subject in need thereof aneffective amount of a composition prepared by a process includingincubating platelets with an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof an effectiveamount of a composition including platelets or platelet derivatives andan incubating agent including one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

Also provided herein are methods of preparing a subject for surgery,including administering to the subject in need thereof an effectiveamount of a composition prepared by a process including incubatingplatelets with an incubating agent including one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent,to form the composition.

In some embodiments of preparing a subject for surgery, the surgery isan emergency surgery.

In some embodiments of preparing a subject for surgery, the surgery is ascheduled surgery.

In some embodiments of any of the methods described herein, the subjecthas been treated or is being treated with an anticoagulant. In someembodiments of any of the methods described herein, treatment with theanticoagulant is stopped. In some embodiments of any of the methodsdescribed herein, treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof an effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent.

Also provided herein are methods of ameliorating the effects of ananticoagulant in a subject, including administering to the subject inneed thereof an effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition.

In some embodiments of ameliorating the effects of an anticoagulant in asubject, the composition is administered following administration to thesubject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, theanticoagulant is selected from the group consisting of dabigatran,argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux,warfarin, heparin, a low molecular weight heparin, a supplement, and acombination thereof. In some embodiments of any of the methods describedherein, the anticoagulant is selected from the group consisting ofdabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban,fondaparinux, warfarin, heparin, low molecular weight heparins,tifacogin, Factor VIIai, SB249417, pegnivacogin (with or withoutanivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban,betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon,acenocoumarol, indandiones, fluindione, a supplement, and a combinationthereof. In some embodiments of any of the methods described herein, theanticoagulant is warfarin. In some embodiments of any of the methodsdescribed herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, before theadministering, the subject had an INR of at least 4.0. In someembodiments of any of the methods described herein, after theadministering, the subject has an INR of 3.0 or less. In someembodiments of any of the methods described herein, after theadministering, the subject has an INR of 2.0 or less. In someembodiments of any of the methods described herein, before theadministering, the subject had an INR of at least 3.0. In someembodiments of any of the methods described herein, after theadministering, the subject has an INR of 2.0 or less.

In some embodiments of any of the methods described herein, the subjecthas been treated or is being treated with an anti-platelet agent. Insome embodiments of any of the methods described herein, treatment withthe antiplatelet agent is stopped. In some embodiments of any of themethods described herein, treatment with the antiplatelet agent iscontinued.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, including administering to the subjectin need thereof an effective amount of a composition including plateletsor platelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent.

Also provided herein are methods of ameliorating the effects of anantiplatelet agent in a subject, including administering to the subjectin need thereof an effective amount of a composition prepared by aprocess including incubating platelets with an incubating agentincluding one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an antiplatelet agentin a subject, the composition is administered following administrationto the subject or assumption by subject, or an overdose of theantiplatelet agent.

In some embodiments of any of the methods described herein, theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, a supplement, and a combination thereof. In some embodimentsof any of the methods described herein, the antiplatelet agent isselected from the group consisting of aspirin, cangrelor, ticagrelor,clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban,picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, anda combination thereof. In some embodiments of any of the methodsdescribed herein, where the antiplatelet agent is selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol,prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium,sarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein, thecomposition includes an anti-fibrinolytic agent. In some embodiments ofany of the methods described herein, the anti-fibrinolytic agent isselected from the group consisting of ε-aminocaproic acid (EACA),tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and acombination thereof. In some embodiments of any of the methods describedherein, the platelets or platelet derivatives are loaded with theanti-fibrinolytic agent.

In some embodiments of any of the methods described herein,administering includes administering topically. In some embodiments ofany of the methods described herein, administering includesadministering parenterally. In some embodiments of any of the methodsdescribed herein, administering includes administering intravenously. Insome embodiments of any of the methods described herein, administeringincludes administering intramuscularly. In some embodiments of any ofthe methods described herein, administering includes administeringintrathecally. In some embodiments of any of the methods describedherein, administering includes administering subcutaneously. In someembodiments of any of the methods described herein, administeringincludes administering intraperitoneally.

In some embodiments of any of the methods described herein, theincubating agent includes a carrier protein. In some embodiments of anyof the methods described herein, the buffer includes HEPES, sodiumbicarbonate (NaHCO₃), or a combination thereof. In some embodiments ofany of the methods described herein, the composition includes one ormore saccharides. In some embodiments of any of the methods describedherein, the one or more saccharides includes trehalose. In someembodiments of any of the methods described herein, the one or moresaccharides includes polysucrose. In some embodiments of any of themethods described herein, the one or more saccharides includes dextrose.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing dose-dependent ε-aminocaproic acid (EACA)loading into platelets at 50 mM and 100 mM using Dansyl-EACA at a molarratio of 1:1000 in the loading buffer in order to measure loading viafluorescence over time at 1 hour, 2 hours, 3 hours, and 4 hours.

FIG. 2 is a graph showing in vitro agonist stimulation of EACA releasefrom EACA-loaded platelets with PMA, collagen, and TRAP agonists usingDansyl-EACA at a molar ratio of 1:1000 in the loading buffer in order tomeasure release via fluorescence.

FIG. 3 is a graph showing an EACA dose-response curve in pooled humanplatelet rich plasma to determine the effect of free EACA on lysis after30 minutes (LY30) via thromboelastography.

FIG. 4 shows that EACA-loaded platelets can release EACA in vitro toprevent fibrinolysis. FIGS. 4A-E show thromboelastogram (TEG) graphs ofEACA loaded platelets with tissue plasminogen activator at varyingplatelet concentrations. FIG. 4F shows a dose-response curve of theeffect of EACA-loaded platelets on LY30.

FIG. 5 is a graph comparing the percent lysis of clots at 30 minutes forfree EACA in solution (FIG. 3) and EACA-loaded platelets (FIG. 4)showing improved response from EACA loaded into platelets.

FIG. 6 is a graph measuring the amount of EACA mg/platelet ofEACA-loaded platelets pre-cryopreservation and post-cryopreservationusing Dansyl-EACA at a molar ratio of 1:1000 in the loading buffer inorder to measure loading via fluorescence.

FIG. 7 shows that cryopreserved EACA-loaded platelets can release EACAin vitro to prevent fibrinolysis. FIGS. 7A-D show TEG graphs ofcryopreserved EACA loaded platelets plus tissue plasminogen activator atvarying platelet concentrations. FIG. 7E shows a dose-response curve ofcryopreserved EACA loaded platelets.

FIGS. 8A-C show graphs indicating the strength of clots as measured bymaximum amplitude (MA). FIG. 8A shows MA measured in the presence offree EACA.

FIGS. 8B-C shows MA measured with EACA loaded plateletspre-cryopreservation (8B) and post-cryopreservation (8C).

FIG. 9 is a graph showing an activated Partial Thromboplastin Time(aPTT) assay on normal plasma with varying concentrations of Factor VIIIneutralizing antibodies. Normal plasma and abnormal plasma controlsincluded in columns 3 and 4, respectively.

FIG. 10 is a graph showing an aPTT assay on normal plasma with varyingconcentrations of Factor IX neutralizing antibodies.

FIG. 11 is a graph showing endogenous thrombin potential (ETP) decreasedwith anti-Factor VIII antibodies in plasma.

FIG. 12 is a histogram of thrombin production in plasma samples withvarying concentrations of Factor VIII antibodies.

FIG. 13 is a graph showing ETP decreased with Factor IX antibodies inplasma.

FIG. 14 is a histogram showing thrombin production in plasma sampleswith varying concentrations of Factor IX antibodies.

FIG. 15 is a histogram showing thrombin generation in platelet richplasma from subject 1 in the presence of Factor VIII antibodies andvarying concentrations of thrombosomes.

FIG. 16 is a graph showing partial recovery of thrombin generation fromplatelet rich from subject 1 in the presence of Factor VIII antibodiesand varying concentrations of thrombosomes.

FIG. 17 is a graph showing return to normal ETP of platelet rich plasmafrom subject 1 in the presence of Factor VIII antibodies and varyingconcentrations of thrombosomes.

FIG. 18 is a histogram showing thrombin generation in platelet richplasma from subject 2 inhibited by Factor VIII antibodies and partiallyrecovered by thrombosomes.

FIG. 19 is a graph showing partial recovery of thrombin generation ofplatelet rich plasma from subject 2 in the presence of factor VIIIantibodies.

FIG. 20 is a graph showing a return to normal ETP of platelet richplasma from subject 2 in the presence of Factor VIII antibodies.

FIG. 21 shows a histogram of thrombin generation in platelet rich plasmafrom subject 1 in the presence of Factor IX antibodies and varyingconcentrations of thrombosomes.

FIG. 22 shows partial recovery of thrombin generation in platelet richplasma from subject 1 in the presence of Factor IX antibodies.

FIG. 23 shows partial recovery to normal thrombin generation potentialof platelet rich plasma from subject 1 in the presence of Factor IXantibodies.

FIG. 24 shows a histogram of thrombin generation in platelet rich plasmafrom subject 2 was inhibited by Factor IX antibodies and partiallyrecovered by the addition of thrombosomes.

FIG. 25 shows partial recovery of thrombin generation of platelet richplasma from subject 2 in the presence of Factor IX antibodies.

FIG. 26 shows a partial return to normal thrombin generation potentialof platelet rich plasma from subject 2 in the presence of Factor IXantibodies.

FIG. 27 shows full recovery of the time to thrombin production by theaddition of thrombosomes to whole blood in the presence of Factor VIIIantibodies

FIG. 28 shows a thromboelastography graph measuring the time to clot (rtime), rate of clot formation (k time), angle of the clot size, and MAsize of the clot.

FIG. 29 is a graph showing time to clot (“r” time) with thrombosomes inthe presence of Factor VIII antibodies.

FIG. 30 is a graph showing time to clot (“r” time) with thrombosomes inthe presence of Factor IX antibodies.

FIG. 31 is a model thromboelastography graph showing model coagulationand fibrinolysis during clot formation.

FIGS. 32A-D show a thromboelastography graphs measuring hemostasisrestoration in platelet rich plasma models of acquired Hemophilia A.

FIGS. 33A-E show thromboelastography graphs measuring hemostasis in aplatelet rich plasma model of acquired Hemophilia B under variousconditions.

FIG. 34 is a graph showing restoration of thrombin generations (nM) in aplatelet rich plasma model of acquired Hemophilia A.

DETAILED DESCRIPTION

This disclosure is directed to compositions and methods for use ofplatelets, platelet derivatives, or thrombosomes as biological carriersof cargo, such as anti-fibrinolytic compounds, also referred to hereinas anti-fibrinolytic loaded platelets, platelet derivatives, orthrombosomes. Also provided herein, are methods of preparing platelets,platelet derivatives, or thrombosomes loaded with anti-fibrinolyticcompounds. This disclosure is also directed to compositions and methodsfor use of unloaded platelets, platelet derivatives, or thrombosomes inthe treatment of a disease such as acquired hemophilia, or conditionssuch as hemorrhaging or trauma.

Anti-fibrinolytic loaded platelets described herein can be stored undertypical ambient conditions, refrigerated, cryopreserved, for examplewith dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization(e.g., to form thrombosomes).

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Further, where a range of values is disclosed, theskilled artisan will understand that all other specific values withinthe disclosed range are inherently disclosed by these values and theranges they represent without the need to disclose each specific valueor range herein. For example, a disclosed range of 1-10 includes 1-9,1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, and so forth. In addition, eachdisclosed range includes up to 5% lower for the lower value of the rangeand up to 5% higher for the higher value of the range. For example, adisclosed range of 4-10 includes 3.8-10.5. This concept is captured inthis document by the term “about”.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a platelet” includes aplurality of such platelets. Furthermore, the use of terms that can bedescribed using equivalent terms include the use of those equivalentterms. Thus, for example, the use of the term “subject” is to beunderstood to include the terms “patient”, “individual,” or “animal” andother terms used in the art to indicate one who is subject to atreatment.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from a disease (e.g., acquired hemophilia),disorder, and/or condition (e.g., hemorrhage) which reduces the severityof the disease, disorder, and/or conditions or slows the progression ofthe disease, disorder, or condition (“therapeutic treatment”), and whichcan inhibit the disease, disorder, and/or condition (e.g., hemorrhage).

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of is an amount sufficient to provide a therapeuticbenefit in the treatment of the disease, disorder and/or condition(e.g., hemorrhage) or to delay or minimize one or more symptomsassociated with the disease, disorder, and/or condition. Atherapeutically effective amount means an amount of therapeutic agent,alone or in combination with other therapies, which provides atherapeutic benefit in the treatment of the disease, disorder, and/orcondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the disease, disorder and/or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the term “platelet” caninclude whole platelets, fragmented platelets, platelet derivatives, orthrombosomes. “Platelets” within the above definition may include, forexample, platelets in whole blood, platelets in plasma, platelets inbuffer optionally supplemented with select plasma proteins, cold storedplatelets, dried platelets, cryopreserved platelets, thawedcryopreserved platelets, rehydrated dried platelets, rehydratedcryopreserved platelets, lyopreserved platelets, thawed lyopreservedplatelets, or rehydrated lyopreserved platelets. “Platelets” may be“platelets” of mammals, such as of humans, or such as of non-humanmammals.

Thus, for example, reference to “anti-fibrinolytic loaded platelets” maybe inclusive of anti-fibrinolytic loaded platelets as well asanti-fibrinolytic loaded platelet derivatives or anti-fibrinolyticloaded thrombosomes, unless the context clearly dictates a particularform.

As used herein, “thrombosomes” (sometimes also herein called “Tsomes” or“Ts”, particularly in the Examples and Figures) are platelet derivativesthat have been treated with an incubating agent (e.g., any of theincubating agents described herein) and lyopreserved (e.g., freeze-driedto form thrombosomes). In some cases, thrombosomes can be prepared frompooled platelets. Thrombosomes can have a shelf life of 2-3 years in dryform at ambient temperature and can be rehydrated with sterile waterwithin minutes for immediate infusion.

As used herein and in the appended claims, the term “fresh platelet”includes platelets stored for less than approximately 24 hours.

As used herein and in the appended claims the term “stored platelet”includes platelets stored for approximately 24 hours or longer beforeuse.

As used herein and in the appended claims the term “fixed platelet”includes platelets fixed with a formalin solution.

As used herein and in the appended claims the term “unloaded” includesplatelets, platelet derivatives, and/or thrombosomes that are not loadedwith an active agent, such as platelets, platelet derivatives, and/orthrombosomes that are not loaded with an anti-fibrinolytic.

In some embodiments, rehydrating the anti-fibrinolytic loaded plateletsincludes adding to the platelets an aqueous liquid. In some embodiments,the aqueous liquid is water. In some embodiments, the aqueous liquid isan aqueous solution. In some embodiments, the aqueous liquid is a salinesolution. In some embodiments, the aqueous liquid is a suspension.

In some embodiments, the rehydrated platelets have coagulation factorlevels showing all individual factors (e.g., Factors VII, VIII and IX)associated with blood clotting at 40 international units (IU) orgreater.

As used herein, “coagulopathy” is a bleeding disorder in which theblood's ability to coagulate (e.g., form clots) is impaired. Thiscondition can cause a tendency toward prolonged or excessive bleed(e.g., diathesis). In some embodiments, a coagulopathy is caused by adisease (e.g., acquired hemophilia). In some embodiments, a coagulopathyis a drug induced coagulopathy. In some embodiments, a coagulopathy isinduced by an antiplatelet agent-induced coagulopathy. In someembodiments, a coagulopathy is induced by an anti-platelet agent.

Accordingly, also provided herein are methods and compositions that aresuitable for treating drug-induced coagulopathy. Anticoagulant drugs,such as warfarin, heparin, and the novel oral anticoagulants (NOACs)class inhibit various plasma factors of the coagulation cascade,resulting in increased bleeding potential.

Anticoagulant drugs are common in the U.S. adult population and employ awide variety of mechanisms to disable segments of the clotting cascade.Anticoagulants are used to treat a number of cardiac or thromboembolicevents. For example, warfarin (e.g., COUMADIN®) is approved for theprophylaxis and treatment of venous thrombosis and its extension,pulmonary embolism; the prophylaxis and treatment of thromboemboliccomplications associated with atrial fibrillation and/or cardiac valvereplacement; the reduction in the risk of death, recurrent myocardialinfarction, and thromboembolic events such as stroke or systemicembolization after myocardial infarction (see, e.g., PrescribingInformation for warfarin (COUMADIN®)). As another example, heparin isapproved for the treatment of thrombophlebitis, phlebothrombosis, andcerebral, coronary, and retinal vessel thrombosis to prevent extensionof clots and thromboembolic phenomena. It is also used prophylacticallyto prevent the occurrence of thromboembolism, and to prevent clottingduring dialysis and surgical procedures, particularly vascular surgery.Other drugs that have anticoagulant properties can include agents thatinhibit factor IIa (thrombin) (also called anti-IIa agents, thrombininhibitors, or direct thrombin inhibitors, depending on the mechanism ofaction), including dabigatran (e.g., PRADAXA®), argatroban, and hirudin;and agents that inhibit factor Xa, including rivaroxaban (e.g.,XARELTO®), apixaban (e.g., ELIQUIS®), edoxaban (e.g., SAVAYSA®), andfondaparinux (e.g., ARIXTRA®). Traditional anticoagulants can includewarfarin (e.g., COUMADIN®) and heparin/LMWH (low molecular weightheparins). Additional anticoagulants include heparainoids, factor IXinhibitors, Factor XI inhibitors, Factor VIIa inhibitors, and TissueFactor inhibitors.

As used herein, an “anticoagulant” is an antithrombotic that does notinclude antiplatelet agents. Examples of antiplatelet agents includeaspirin, cangrelor, ticagrelor, clopidogrel (e.g., PLAVIX®), prasugreleptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), andabciximab (e.g., REOPRO®). Typically, agents that inhibit P2Y receptors(e.g., P2Y12), glycoprotein IIb/IIIa, or that antagonize thromboxanesynthase or thromboxane receptors, are considered to be antiplateletagents. Other mechanisms of antiplatelet agents are known. As usedherein, aspirin is considered to be an antiplatelet agent but not ananticoagulant.

Agents that inhibit Factor IIa, VIIa, IX, Xa, XI, Tissue Factor, orvitamin K-dependent synthesis of clotting factors (e.g., Factor II, VII,IX, or X) or that activate antithrombin (e.g., antithrombin III) areanticoagulants for the purpose of the present disclosure. Othermechanisms of anticoagulants are known. Non-limiting examples ofanticoagulants include dabigatran, argatroban, hirudin, rivaroxaban,apixaban, edoxaban, fondaparinux, warfarin, heparin, and low molecularweight heparins (e.g., dalteparin, enoxaparin, tinzaparin, ardeparin,nadroparin, reveparin, danaparoid). Additional non-limiting examples ofanticoagulants include tifacogin, Factor VIIai, SB249417, pegnivacogin(with or without anivamersen), TTP889, idraparinux, idrabiotaparinux,SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran,phenprocoumon, acenocoumarol, indandiones, and fluindione. In someembodiments, the anticoagulant is selected from the group consisting ofdabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban,fondaparinux, warfarin, heparin, low molecular weight heparins,tifacogin, Factor VIIai, SB249417, pegnivacogin (with or withoutanivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban,betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon,acenocoumarol, indandiones, and fluindione.

Overcoming the effect of an anticoagulant varies according to theanticoagulant drug pharmacological action. In the case of advancednotice, as in a pre-planned surgery, the anti-coagulant dose cansometimes be tailored back before the surgery; however, there may becases where such a reduction in dose is not advisable. In the case wherean anti-coagulant need reversing quickly (e.g., for emergency surgery),reversal agents are typically slow acting, expensive, or carrysignificant risk to the patient.

Warfarin (e.g., COUMADIN®)—Warfarin works to prevent the activity ofvitamin K in the liver which is a necessary co-factor to producemultiple coagulation factors. Warfarin reversal can sometimes be done beby dosing vitamin K or prothrombin complex concentrate (PCC). Vitamin Kis low-cost and slow acting (more than 24 hrs PO) but can posesignificant risk of inducing thrombosis in the patient, while PCC isexpensive at roughly $5000/dose.

Dabigatran (e.g., PRADAXA®)—Dabigatran is a direct inhibitor ofthrombin. The monoclonal antibody therapy idarucizumab (e.g., PRAXBIND®,Boehringer-Ingelheim, Germany) at dose of 5 grams (at two dose intervalseach 2.5 grams) can typically reverse the effects of dabigatran within afew minutes. One wholesale price is $3482.50 for such a treatment.

Rivaroxaban (e.g., XARELTO®)—Rivaroxaban is a direct Factor Xainhibitor. Rivaroxaban is reversed by Andexanet Alfa (e.g., ANDEXXA®), arecombinant Factor Xa decoy. This treatment can cost roughly $50,000 fora high-dose treatment.

Apixaban (e.g., ELIQUIS®)—Apixaban is a direct Factor Xa inhibitor.Apixaban is reversed by Andexanet Alfa, a recombinant Factor Xa decoy.This treatment costs roughly can cost $50,000 for a high-dose treatment.

Edoxaban (e.g., SAVAYSA®, LIXIANA®)—Edoxaban is a direct Factor Xainhibitor. Exoxaban does not have an approved reversal agent.Ciraparantag (aripazine) and Andexanet Alfa have not been clinicallyproven to be appropriate.

Heparin and low molecular weight heparins are activators of antithrombinIII (AT). AT inactivates proteases such as thrombin and Factor Xa.Protamine sulfate is a highly positively-charged polypeptide that bindsto the negatively charged heparin and prevents its action on AT.Protamine sulfate is typically dosed at about 1.0 to about 1.5 mg/100 IUof active heparin.

Platelet-derived products (e.g., thrombosomes, cryo-preserved plateletsare not currently used as a treatment method for anticoagulant drugs. Insome embodiments, platelet derived products, including anti-fibrinolyticloaded platelets and anti-fibrinolytic loaded thrombosomes (e.g.,freeze-dried platelets) are used to as a treatment method foranticoagulant drugs.

Treatments for anticoagulant drugs are not necessarily targetedantidotes. Some novel anticoagulant treatments, such as Andexanet Alfa(e.g., ANDEXXA®), have seen some success, yet can be expensive. As such,emergency treatments (pre-op, trauma, and the like) are typicallyblanket precautions to avoid or mitigate hemorrhage. Non-limitingexamples include infusion of plasma, red blood cells, andanti-fibrinolytics. Products and methods are described herein forcontrolling bleeding and improving healing. The products and methodsdescribed herein can also be used to counteract the activity of ananticoagulant.

Products and methods are described herein for controlling bleeding andimproving healing. The products and methods described herein can also beused to counteract the activity of an anticoagulant (e.g., warfarin(e.g., COUMADIN®), heparin, LMWH, dabigatran (e.g., PRADAXA®),argatroban, hirudin, rivaroxaban (e.g., XARELTO®), apixaban (e.g.,ELIQUIS®), edoxaban (e.g., SAVAYSA®), fondaparinux (e.g., ARIXTRA®). Theproducts and methods described herein are directed toward embodimentsthat aid in the closure and healing of wounds.

In certain embodiments, a composition comprising platelets such aslyophilized platelets or platelet derivatives may be delivered to awound on the surface of or in the interior of a patient. In variousembodiments, a composition comprising platelets or platelet derivativescan be applied in selected forms including, but not limited to, adhesivebandages, compression bandages, liquid solutions, aerosols, matrixcompositions, and coated sutures or other medical closures. In someembodiments, a platelet derivative may be administered to all or only aportion of an affected area on the surface of a patient. In otherembodiments, a composition comprising platelets such as lyophilizedplatelets or platelet derivatives may be administered systemically, forexample via the blood stream. In embodiments, an application of theanti-fibrinolytic loaded platelet derivative can produce hemostaticeffects for 2 or 3 days, preferably 5 to 10 days, or most preferably forup to 14 days.

As used herein, an “antiplatelet agent” is an antithrombotic and doesnot include anticoagulants. Examples of antiplatelet agents includeaspirin (also called acetylsalicylic acid or ASA), cangrelor (e.g.,KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®),prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban(e.g., AGGRASTAT®), and abciximab (e.g., REOPRO®). For the purpose ofthis disclosure, antiplatelet agents include agents that inhibit P2Yreceptors (e.g., P2Y12), glycoprotein IIb/IIIa, or that antagonizethromboxane synthase or thromboxane receptors. Non-limiting examples ofthromboxane A2 antagonists are aspirin, terutroban, and picotamide.Non-limiting examples of P2Y receptor antagonists include cangrelor,ticagrelor, elinogrel, clopidogrel, prasugrel, and ticlopidine.Non-limiting examples of glycoprotein IIb/IIIa include abciximab,eptifibatide, and tirofiban. NSAIDS (e.g., ibuprofen) are alsoconsidered to be antiplatelet agents for the purposes of thisdisclosure. Other mechanisms of anti-platelet agents are known.Antiplatelet agents also include PARI antagonists, PAR4 antagonists GPVIantagonists and alpha2beta1 collagen receptor antagonists. Non-limitingexamples of PAR-1 antagonists include vorapaxar and atopaxar. As usedherein, aspirin is considered to be an antiplatelet agent but not ananticoagulant. Additional non-limiting examples of antiplatelet agentsinclude cilostazol, prostaglandin E1, epoprostenol, dipyridamole,treprostinil sodium, and sarpogrelate.

In some embodiments, an antiplatelet agent can be selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, and combinations thereof.In some embodiments, an antiplatelet agent can be selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and combinationsthereof. In some embodiments, an antiplatelet agent can be selected fromthe group consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol,prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium,sarpogrelate and combinations thereof. In some embodiments, theantiplatelet agent can include multiple antiplatelet agents, such as 2(or more) of any of the antiplatelet agents described herein. In someembodiments, the antiplatelet agent can be aspirin and clopidogrel.

Cangrelor like clopidogrel, ticagrelor, and prasugrel, blocks the P2Y12(ADP) receptor on platelets. Cangrelor can in some cases be used as arepresentative of this class of drug. Cangrelor, unlike clopidogrel andprasugrel, does not need hepatic metabolism to become biologicallyactive.

Eptifibatide is a peptide therapeutic that blocks the fibrin bindingrole of GPIIb-IIIa receptor on platelets. The drug is typicallyadministered via IV as a 180 μg/kg bolus followed by 2 μg/kg/mincontinuous infusion. The blood concentration of eptifibatide istypically about 1-2 μM. Bleeding times generally return to normal withinabout 1 hour of drug stoppage.

Aspirin is an irreversible cylcooxygenase (COX) inhibitor. The COXenzyme in platelets is responsible for synthesis of thromboxane A2,prostaglandin E2 and prostacyclin (PGI2). Aspirin permanentlyinactivates the COX enzyme within platelets, and since platelets do nothave the nuclear material to synthesize new enzyme, new platelets mustbe produced to overcome the aspirin effect. Without thromboxane A2,prostaglandin E2, and prostacyclin (PGI2) platelets are limited in theirpro-aggregation activity. Many people are maintained on a low dose ofaspirin to prevent unwanted clotting events. Aspirin bioavailabilitylargely varies with administration route, with a single 500 mg dose IVat peaks of 500 μM and the same dose orally at 44 μM.

The antiplatelet class of drugs is widely used to prevent unwantedclotting episodes that lead to heart failure, stroke, and the like. Inmany cases, an antiplatelet drug may need to be reversed or stopped. Inthe case of advanced notice, as in a pre-planned surgery situation, theantiplatelet drug dose can sometimes be stopped before the surgery,preventing unwanted bleeding during surgery. In the case where anantiplatelet agent needs reversing quickly, reversal agents aretypically not readily available, are expensive, or carry significantrisk to the patient. In the case of need for rapid antiplateletreversal, a platelet transfusion is typically administered, though theresponse to this is often only partial reversal. The caveat of thiscourse of reversal is that the newly-infused platelets themselves aresusceptible to circulating drug antiplatelet activity whereas, in someembodiments, compositions as described herein (e.g., includingthrombosomes) are not. In some embodiments, compositions as describedherein (e.g., including thrombosomes) are an active reversal agent. Insome embodiments, the hemostatic activity of compositions as describedherein (e.g., including thrombosomes) does not succumb to antiplateletdrugs.

Some exemplary antiplatelet agents and potential methods of reversal aredescribed below.

Acetylsalicylic acid (ASA; aspirin)—aspirin acts as a COX-1 blocker inplatelets, which renders the platelet inactive by irreversiblyinhibiting platelet-derived thromboxane formation. Clinically, aspirinis sometimes reversed by a platelet transfusion in emergency situationsor by stopping treatment where surgery is scheduled in the future.

Clopidogrel (e.g., PLAVIX®)—clopidogrel acts as to prevent ADP frombinding to its receptor on platelets. ADP binding leads to plateletshape change and aggregation. Clopidogrel is non-reversible. Clinically,clopidogrel is sometimes reversed by a platelet transfusion in emergencysituations or by stopping treatment where surgery is scheduled in thefuture.

Cangrelor (e.g., KENGREAL®)—cangrelor acts to prevent ADP from bindingto its receptor on platelets. ADP binding leads to platelet shape changeand aggregation. Clopidogrel is reversible and platelet function isreturned approximately 1 hour after stopping infusion. Clinically it isgenerally preferred when reversal is needed after a procedure.

Ticagrelor (e.g., BRILINTA®)—ticagrelor acts to prevent ADP from bindingto its receptor and acts as an inverse agonist. Ticagrelor is reversibleand platelet function can return after approximately 72 hours of thelast dosage. Reversal of action of ticagrelor can be affected by thetime after the last dose. If the last dose was longer than 24 hoursprevious, then platelet transfusion can sometimes be therapeutic toreverse the results.

Effient (e.g., PRASUGREL®)—Effient acts to prevent ADP from binding toits receptor and acts as a non-reversable antagonist. It being anon-reversible antagonist, new platelets must be formed to overcomingits effect. Clinically Effient is reversed by a platelet transfusion inemergency situations or by stopping treatment where surgery is scheduledin the future.

Eptifibatide (Integrilin)—Eptifibatide acts to block the GpIIb/IIIa andacts as a reversible antagonist. Clinically, Integrilin is reversed by aplatelet transfusion in emergency situations or by stopping treatmentwhere surgery is scheduled in the future.

Platelet-derived products are not currently used as a treatment methodfor anticoagulant/antiplatelet drugs, and there are no currentlyapproved reversal agents for antiplatelet agents. As such, emergencytreatments (pre-op, trauma, and the like) are typically blanketprecautions to avoid or mitigate hemorrhage. Non-limiting examplesinclude infusion of plasma, red blood cells, and anti-fibrinolytics.Platelet derivatives (e.g., lyopreserved platelets (thrombosomes)) maybe an effective alternative or supplement to these general treatments.

Products and methods are described herein for controlling bleeding andimproving healing. The products and methods described herein can also beused to counteract the activity of an antiplatelet agent (e.g., aspirin(also called acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®),ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel(e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g.,AGGRASTAT®), or abciximab (e.g., REOPRO®)). The products and methodsdescribed herein are directed toward embodiments that can aid in theclosure and healing of wounds.

Unless specified as loaded, platelets (e.g., freeze-dried (e.g.,lyophilized) platelets), or platelet derivatives may or may not have notbeen loaded with a therapeutic agent (e.g., an anti-fibrinolytic). Thus,for example, the following: in some embodiments, platelets (e.g.,anti-fibrinolytic loaded platelets), lyophilized platelets (e.g.,anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives(e.g., anti-fibrinolytic loaded platelet derivatives) are used to treata coagulopathy contemplates both the use of unloaded platelets, unloadedlyophilized platelets, or unloaded platelet derivatives and the use ofanti-fibrinolytic loaded platelets, anti-fibrinolytic loaded lyophilizedplatelets, or anti-fibrinolytic loaded platelet derivatives, or acombination thereof to treat a coagulopathy.

In some embodiments, the dried platelets, such as freeze-driedplatelets, have less than about 10%, such as less than about 8%, such asless than about 6%, such as less than about 4%, such as less than about2%, such as less than about 0.5% crosslinking of platelet membranes viaproteins and/or lipids present on the membranes. In some embodiments,the dried platelets, such as freeze dried platelets, have less thanabout 10%, such as less than about 8%, such as less than about 6%, suchas less than about 4%, such as less than about 2%, such as less thanabout 0.5% crosslinking of platelet membranes via proteins and/or lipidspresent on the membranes. In some embodiments, the rehydrated platelets,have less than about 10%, such as less than about 8%, such as less thanabout 6%, such as less than about 4%, such as less than about 2%, suchas less than about 0.5% crosslinking of platelet membranes via proteinsand/or lipids present on the membranes. In some embodiments, therehydrated platelets, have between about 0.01% to about 5%, such asbetween about 0.1% to about 4%, such as between about 1% to betweenabout 3%, such as between about 1% to about 2%, crosslinking of plateletmembranes via proteins and/or lipids present on the membranes. In someembodiments, the rehydrated platelets, have at least about 1% to atleast about 10, such as less than about 8%, such as less than about 6%,such as less than about 4%, such as less than about 2%, such as lessthan about 0.5% crosslinking of platelet membranes via proteins and/orlipids present on the membranes.

In some embodiments, the anti-fibrinolytic loaded platelets and thedried platelets, such as freeze-dried platelets, having a particle size(e.g., diameter, max dimension) of at least about 0.2 μm (e.g., at leastabout 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at leastabout 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at leastabout 0.9 μm, at least about 1.0 μm, at least about 1.0 μm, at leastabout 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at leastabout 5.0 μm). In some embodiments, the particle size is less than about5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less thanabout 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less thanabout 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less thanabout 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). Insome embodiments, the particle size is from about 0.3 μm to about 5.0 μm(e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8μm).

In some embodiments, at least 50% (e.g., at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99%) of platelets and/or the driedplatelets, such as freeze-dried platelets, have a particle size in therange of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about0.9 μm, or from about 0.6 μm to about 0.8 μm). In some embodiments, atmost 99% (e.g., at most about 95%, at most about 80%, at most about 75%,at most about 70%, at most about 65%, at most about 60%, at most about55%, or at most about 50%) of platelets and/or the dried platelets, suchas freeze-dried platelets, are in the range of about 0.3 μm to about 5.0μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about0.8 μm). In some embodiments, about 50% to about 99% (e.g., about 55% toabout 95%, about 60% to about 90%, about 65% to about 85, about 70% toabout 80%) of platelets and/or the dried platelets, such as freeze-driedplatelets, are in the range of about 0.3 μm to about 5.0 μm (e.g., fromabout 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, fromabout 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, fromabout 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, (e.g., using unloaded platelets or plateletderivatives), the platelets or platelet derivatives are preparedconsistent with the procedures described in U.S. Pat. No. 8,486,617(such as, e.g., Examples 1-5) and U.S. Pat. No. 8,097,403 (such as,e.g., Examples 1-3).

Also provided herein are methods of preparing anti-fibrinolytic loadedplatelets. In some embodiments, platelets are isolated prior tocontacting the platelets with an anti-fibrinolytic.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; and step (b) contacting theplatelets with an anti-fibrinolytic, and with a loading buffercomprising a salt, a base, a loading agent, and optionally ethanol, toform the anti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; step (b) contacting theplatelets with an anti-fibrinolytic to form a first composition; andstep (c) contacting the first composition with a buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent to form the anti-fibrinolytic loaded platelets.

In some embodiments, suitable organic solvents include, but are notlimited to alcohols, esters, ketones, ethers, halogenated solvents,hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixturesthereof. In some embodiments, suitable organic solvents includes, butare not limited to methanol, ethanol, n-propanol, isopropanol, aceticacid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methylacetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropylether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane),acetonitrile, propionitrile, methylene chloride, chloroform, toluene,anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane,dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone,dimethylacetamide, and combinations thereof.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; step (b) contacting theplatelets with a buffer comprising a salt, a base, a loading agent, andoptionally at least one organic solvent, to form a first composition;and step (c) contacting the first composition with an anti-fibrinolytic,to form the anti-fibrinolytic loaded platelets.

In some embodiments, isolating platelets includes isolating plateletsfrom blood.

In some embodiments, platelets are donor-derived platelets. In someembodiments, platelets are obtained by a process that includes anapheresis step. In some embodiments, platelets are fresh platelets. Insome embodiments, platelets are stored platelets.

In some embodiments, platelets are derived in vitro. In someembodiments, platelets are derived or prepared in a culture prior tocontacting the platelets with an anti-fibrinolytic. In some embodiments,preparing the platelets includes deriving or growing the platelets froma culture of megakaryocytes. In some embodiments, preparing theplatelets includes deriving or growing the platelets (or megakaryocytes)from a culture of human pluripotent stem cells (PCSs), includingembryonic stem cells (ESCs) and/or induced pluripotent stem cells(iPSCs).

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) preparingplatelets; and step (b) contacting the platelets with ananti-fibrinolytic and with a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent, to form theanti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) preparingplatelets; step (b) contacting the platelets with an anti-fibrinolyticto form a first composition; and step (c) contacting the firstcomposition with a buffer comprising a salt, a base, a loading agent,and optionally at least one organic solvent, to form theanti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) preparingplatelets; step (b) contacting the platelets with a buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form a first composition; and step (c) contacting the firstcomposition with an anti-fibrinolytic, to form the anti-fibrinolyticloaded platelets.

In some embodiments, no solvent is used. Thus, in some embodiments, themethod for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium;        -   and    -   b) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets,        -   wherein the method does not comprise contacting the            platelets with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolyticloaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium;    -   b) contacting the platelets with an anti-fibrinolytic to form a        first composition; and    -   c) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the anti-fibrinolytic        loaded platelets, wherein the method does not comprise        contacting the platelets with an organic solvent such as ethanol        and the method does not comprise contacting the first        composition with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolyticloaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium;    -   b) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition; and    -   c) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets.        -   wherein the method does not comprise contacting the            platelets with an organic solvent such as ethanol and the            method does not comprise contacting the first composition            with an organic solvent such as ethanol.

In some embodiments, the method for preparing anti-fibrinolytic loadedplatelets comprises:

-   -   a) preparing platelets;        -   and    -   b) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets,        -   wherein the method does not comprise contacting the            platelets with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolyticloaded platelets comprises:

-   -   a) preparing platelets;    -   b) contacting the platelets with an anti-fibrinolytic to form a        first composition; and    -   c) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the anti-fibrinolytic        loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolyticloaded platelets comprises:

-   -   a) preparing platelets;    -   b) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition; and    -   c) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets.    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol.

In some embodiments, the loading agent is a saccharide. In someembodiments, the saccharide is a monosaccharide. In some embodiments,the saccharide is a disaccharide. In some embodiments, the saccharide isa non-reducing disaccharide. In some embodiments, the saccharide issucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, orxylose. In some embodiments, the loading agent is a starch. In someembodiments, a loading agent is a cryoprotectant. In some embodiments,(e.g., for platelets or platelet derivatives not loaded with ananti-fibrinolytic agent), a “loading agent” can be used in thepreparation of the platelets or platelet derivatives, for example, aspart of an incubating agent.

As used herein, the term “anti-fibrinolytic,” “anti-fibrinolytics,” or“anti-fibrinolytic compound,” is any compound capable of inhibitingfibrinolysis. Fibrinolysis is the process where the activatedplasminogen removes excess fibrin and promotes fibrin clot formation andwound healing (Szekely, A. and Lex, D. J., Antifibrinolytics, Heart LungVessel, 6(1): 5-7, (2014), which is incorporated herein by reference inits entirety). Inhibiting fibrinolysis can be useful under certainconditions. For example, in the case of traumatic bleeding events and/orhemorrhage, inhibiting fibrinolysis can enhance the formation of bloodclots (e.g., stopping bleeding).

In some embodiments, the anti-fibrinolytic can be ε-aminocaproic acid.In some embodiments, the anti-fibrinolytic can be tranexamic acid. Insome embodiments, the anti-fibrinolytic can be aprotinin. In someembodiments, the anti-fibrinolytic can be aminomethylbenzoic acid. Insome embodiments, the anti-fibrinolytic can be fibrinogen. In someembodiments, the anti-fibrinolytic can be a combination of two or moreanti-fibrinolytics.

In some embodiments, an anti-fibrinolytic (e.g., EACA) loaded intoplatelets is modified to include an imaging agent. For example, ananti-fibrinolytic can be modified with an imaging agent in order toimage the anti-fibrinolytic loaded platelet in vivo. In someembodiments, an anti-fibrinolytic can be modified with two or moreimaging agents (e.g., any two or more of the imaging agents describedherein). In some embodiments, an anti-fibrinolytic loaded into plateletsis modified with a radioactive metal ion, a paramagnetic metal ion, agamma-emitting radioactive halogen, a positron-emitting radioactivenon-metal, a hyperpolarized NMR-active nucleus, a reporter suitable forin vivo optical imaging, or a beta-emitter suitable for intravasculardetection. For example, a radioactive metal ion can include, but is notlimited to, positron emitters such as ⁵⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ⁹⁴Tc or⁶⁸Ga; or gamma-emitters such as ¹⁷¹Tc, ¹¹¹In, ¹¹³In, or ⁶⁷Ga. Forexample, a paramagnetic metal ion can include, but is not limited toGd(III), a Mn(II), a Cu(II), a Cr(III), a Fe(III), a Co(II), a Er(II), aNi(II), a Eu(III) or a Dy(III), an element comprising an Fe element, aneodymium iron oxide (NdFeO3) or a dysprosium iron oxide (DyFeO3). Forexample, a paramagnetic metal ion can be chelated to a polypeptide or amonocrystalline nanoparticle. For example, a gamma-emitting radioactivehalogen can include, but is not limited to ¹²³I, ¹³¹I or ⁷⁷Br. Forexample, a positron-emitting radioactive non-metal can include, but isnot limited to ¹¹C, ¹³N, ¹⁵O, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ¹²⁴I. For example,a hyperpolarized NMR-active nucleus can include, but is not limited to¹³C, ¹⁵N, ¹⁹F, ²⁹Si and ³¹P. For example, a reporter suitable for invivo optical imaging can include, but is not limited to any moietycapable of detection either directly or indirectly in an optical imagingprocedure. For example, the reporter suitable for in vivo opticalimaging can be a light scatterer (e.g., a colored or uncoloredparticle), a light absorber or a light emitter. For example, thereporter can be any reporter that interacts with light in theelectromagnetic spectrum with wavelengths from the ultraviolet to thenear infrared. For example, organic chromophoric and fluorophoricreporters include groups having an extensive delocalized electronsystem, e.g. cyanines, merocyanines, indocyanines, phthalocyanines,naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes,thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes,indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes,anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecularcharge-transfer dyes and dye complexes, tropones, tetrazines,b/s(dithiolene) complexes, bts(benzene-dithiolate) complexes,iodoaniline dyes, b/stS.O-dithiolene) complexes. For example, thereporter can be, but is not limited to a fluorescent, a bioluminescent,or chemiluminescent polypeptide. For example, a fluorescent orchemiluminescent polypeptide is a green florescent protein (GFP), amodified GFP to have different absorption/emission properties, aluciferase, an aequorin, an obelin, a mnemiopsin, a berovin, or aphenanthridinium ester. For example, a reporter can be, but is notlimited to rare earth metals (e.g., europium, samarium, terbium, ordysprosium), or fluorescent nanocrystals (e.g., quantum dots). Forexample, a reporter may be a chromophore that can include, but is notlimited to fluorescein, sulforhodamine 101 (Texas Red), rhodamine B,rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green514, tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, AlexaFluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700, and Alexa Fluor 750. For example, abeta-emitter can include, but is not limited to radio metals ⁶⁷Cu, ⁸⁹Sr,⁹⁰Y, ¹⁵³Sm, ¹⁸⁵Re, ¹⁸⁸Re or ¹⁹²Ir, and non-metals ³²P, ³³P, ³⁸S, ³⁸Cl,³⁹Cl, ⁸²Br and ⁸³Br. In some embodiments, an anti-fibrinolytic loadedinto platelets can be associated with gold or other equivalent metalparticles (such as nanoparticles). For example, a metal particle systemcan include, but is not limited to gold nanoparticles (e.g., Nanogold™).

In some embodiments, an anti-fibrinolytic loaded into platelets that ismodified with an imaging agent is imaged using an imaging unit. Theimaging unit can be configured to image the anti-fibrinolytic loadedplatelets in vivo based on an expected property (e.g., optical propertyfrom the imaging agent) to be characterized. For example, imagingtechniques (in vivo imaging using an imaging unit) that can be used, butare not limited to are: computer assisted tomography (CAT), magneticresonance spectroscopy (MRS), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), or bioluminescence imaging (BLI). Chen, Z., et al., Advance ofMolecular Imaging Technology and Targeted Imaging Agent in Imaging andTherapy, Biomed Res Int., 819324, doi: 10.1155/2014/819324 (2014) havedescribed various imaging techniques and which is incorporated byreference herein in its entirety.

For example, a modified anti-fibrinolytic can be modified such that themodifying group interacts with the anti-fibrinolytic. In a non-limitingway a modifying agent such as dansyl chloride can interact with theanti-fibrinolytic. Dansyl chloride can interact with primary aminogroups in aliphatic and aromatic amines and can produce blue orblue-green sulfonamide adducts. In some embodiments, dansyl chloride caninteract with EACA to generate a modified anti-fibrinolytic (e.g.,dansyl-EACA).

In some embodiments, such as embodiments wherein the platelets aretreated with the an anti-fibrinolytic (e.g., EACA) and the buffersequentially as disclosed herein, the anti-fibrinolytic can be loaded ina liquid medium that can be modified to change the proportion of mediacomponents or to exchange components for similar products, or to addcomponents necessary for a given application.

In some embodiments, the loading buffer and/or the liquid medium includeone or more of a) water or a saline solution, b) one or more additionalsalts, or c) a base. In some embodiments, the loading buffer, and/or theliquid medium, may include one or more of a) DMSO, b) one or more salts,or c) a base.

In some embodiments, the loading agent is loaded into the platelets inthe presence of an aqueous medium. In some embodiments, the loadingagent is loaded in the presence of a medium comprising DMSO. As anexample, one embodiment of the methods herein includes contactingplatelets with an anti-fibrinolytic and with an aqueous loading buffercomprising a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets. As anexample, one embodiment of the methods herein includes contactingplatelets with an anti-fibrinolytic and with a loading buffer comprisingDMSO and comprising a salt, a base, a loading agent, and optionallyethanol, to form the anti-fibrinolytic loaded platelets.

In some embodiments, the loading buffer and/or the liquid medium,include one or more salts selected from phosphate salts, sodium salts,potassium salts, calcium salts, magnesium salts, and any other salt thatcan be found in blood or blood products, or that is known to be usefulin drying platelets, or any combination of two or more of these.

Preferably, these salts are present in the composition at an amount thatis about the same as is found in whole blood.

In some embodiments, the loading buffer and/or liquid medium furthercomprises a carrier protein. In some embodiments, the carrier proteincomprises human serum albumin, bovine serum albumin, or a combinationthereof. In some embodiments, the carrier protein is present in anamount of about 0.05% to about 1.0% (w/v).

In some embodiments, the anti-fibrinolytic loaded platelets are preparedby incubating the platelets with the anti-fibrinolytic in the liquidmedium for different durations at or at different temperatures fromabout 15-45° C., or about 37° C. The step of incubating the platelets toload one or more anti-fibrinolytic compounds includes incubating theplatelets for a time suitable for loading, as long as the time, taken inconjunction with the temperature, is sufficient for theanti-fibrinolytic to come into contact with the platelets and,preferably, be incorporated, at least to some extent, into theplatelets. In some embodiments, the anti-fibrinolytic loaded plateletsare prepared by incubating the platelets with the anti-fibrinolytic inthe liquid medium at a temperature from about 18-42° C., about 20-40°C., about 22-37° C., or about 16° C., about 18° C., about 20° C., about22° C., about 24° C., about 26° C., about 28° C., about 30° C., about32° C., about 34° C., about 36° C., about 37° C., about 39° C., about41° C., about 43° C., or about 45° C. for at least about 5 minutes(mins) (e.g., at least about 20 mins, about 30 mins, about 1 hour (hr),about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs,about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, about 42 hrs,about 48 hrs, or at least about 48 hrs. In some embodiments, theanti-fibrinolytic loaded platelets are prepared by incubating theplatelets with the anti-fibrinolytic in the liquid medium at atemperature from about 18-42° C., about 20-40° C., about 22-37° C., orabout 16° C., about 18° C., about 20° C., about 22° C., about 24° C.,about 26° C., about 28° C., about 30° C., about 32° C., about 34° C.,about 36° C., about 37° C., about 39° C., about 41° C., about 43° C., orabout 45° C. for no more than about 48 hrs (e.g., no more than about 20mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs,about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs,about 30 hrs, about 36 hrs, or no more than about 42 hrs). In someembodiments, the anti-fibrinolytic loaded platelets are prepared byincubating the platelets with the anti-fibrinolytic in the liquid mediumfrom about 10 mins to about 48 hours (e.g., from about 20 mins to about36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20hrs, from about 2 hrs to about 16 hours, from about 10 mins to about 24hours, from about 20 mins to about 12 hours, from about 30 mins to about10 hrs, or from about 1 hr to about 6 hrs.

In one embodiment, contacting platelets with an anti-fibrinolyticincludes contacting the platelets with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent for a period of time, such as a period of 1 minute to 48 hours,such as 2 hours.

In some embodiments, the platelets are at a concentration from about1,000 platelets/μl to about 10,000,000 platelets/μl. In someembodiments, the platelets are at a concentration from about 50,000platelets/μl to about 4,000,000 platelets/μl. In some embodiments, theplatelets are at a concentration from about 100,000 platelets/μl toabout 300,000,000 platelets/μl. In some embodiments, the platelets areat a concentration from about 1,000,000 to about 2,000,000. In someembodiments, the platelets are at a concentration of about 200,000,000platelets/μl.

In some embodiments of the methods of preparing anti-fibrinolytic loadedplatelets disclosed herein, the methods further include acidifying theplatelets, or pooled platelets, to a pH of about 6.0 to about 7.4, priorto a contacting step disclosed herein. In some embodiments, the methodsinclude acidifying the platelets to a pH of about 6.5 to about 6.9. Insome embodiments, the methods include acidifying the platelets to a pHof about 6.6 to about 6.8. In some embodiments, the acidifying includesadding to the pooled platelets a solution comprising Acid CitrateDextrose.

In some embodiments, the platelets are isolated prior to a contactingstep. In some embodiments, the methods further include isolatingplatelets by using centrifugation. In some embodiments, thecentrifugation occurs at a relative centrifugal force (RCF) of about 800g to about 2000 g. In some embodiments, the centrifugation occurs atrelative centrifugal force (RCF) of about 1300 g to about 1800 g. Insome embodiments, the centrifugation occurs at relative centrifugalforce (RCF) of about 1500 g. In some embodiments, the centrifugationoccurs for about 1 minute to about 60 minutes. In some embodiments, thecentrifugation occurs for about 10 minutes to about 30 minutes. In someembodiments, the centrifugation occurs for about 20 minutes.

In some embodiments, the platelets are at a concentration from about1,000 platelets/μl to about 10,000,000 platelets/μl. In someembodiments, the platelets are at a concentration from about 50,000platelets/μl to about 4,000,000 platelets/μl. In some embodiments, theplatelets are at a concentration from about 100,000 platelets/μl toabout 300,000,000 platelets/μl. In some embodiments, the platelets areat a concentration from about 1,000,000 to about 2,000,000. In someembodiments, the platelets are at a concentration of about 2,000,000platelets/μl.

In some embodiments, the buffer is a loading buffer comprising thecomponents as listed in Table 1 herein. In some embodiments, a loadingbuffer is an incubating agent. In some embodiments, the loading bufferincludes one or more salts, such as phosphate salts, sodium salts,potassium salts, calcium salts, magnesium salts, and any other salt thatcan be found in blood or blood products. Exemplary salts include sodiumchloride (NaCl), potassium chloride (KCl), and combinations thereof. Insome embodiments, the loading buffer includes from about 0.5 mM to about100 mM of the one or more salts. In some embodiments, the loading bufferincludes from about 1 mM to about 100 mM (e.g., about 2 mM to about 90mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mMto about 90 mM, about 70 to about 85 mM) about of the one or more salts.In some embodiments, the loading buffer includes about 5 mM, about 75mM, or about 80 mM of the one or more salts.

In some embodiments, the loading buffer includes one or more buffers,e.g., N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),and/or sodium-bicarbonate (NaHCO₃). In some embodiments, the loadingbuffer includes from about 5 to about 100 mM of the one or more buffers.In some embodiments, the loading buffer includes from about 5 to about50 mM (e.g., from about 5 mM to about 40 mM, from about 8 mM to about 30mM, about 10 mM to about 25 mM) about of the one or more buffers. Insome embodiments, the loading buffer includes about 10 mM, about 20 mM,about 25 mM, or about 30 mM of the one or more buffers.

In some embodiments, the loading buffer includes one or moresaccharides, such as monosaccharides and disaccharides, includingsucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. Insome embodiments, the loading buffer includes from about 10 mM to about1,000 mM of the one or more saccharides. In some embodiments, theloading buffer includes from about 50 to about 500 mM of the one or moresaccharides. In embodiments, one or more saccharides is present in anamount of from 10 mM 10 to 500 mM. In some embodiments, one or moresaccharides is present in an amount of from 50 mM to 200 mM. Inembodiments, one or more saccharides is present in an amount from 100 mMto 150 mM.

In some embodiments, the anti-fibrinolytic includes oneanti-fibrinolytic. In some embodiments, the anti-fibrinolytic includestwo or more anti-fibrinolytics.

In some embodiments, the methods further include incubating theanti-fibrinolytic (e.g., EACA) in the presence of the loading bufferprior to the treatment step. In some embodiments, the methods furtherinclude incubating the loading buffer and a solution comprising theanti-fibrinolytic and water at about 37° C. using different incubationperiods. In some embodiments, the solution includes a concentration ofabout 1 μM to about 100 mM of the anti-fibrinolytic. In someembodiments, the solution includes a concentration of about 10 μM toabout 10 mM of the anti-fibrinolytic. In some embodiments, the solutionincludes a concentration of about 100 μM to about 100 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 200 μM to about 1 mM of the anti-fibrinolytic. Insome embodiments, the solution includes a concentration of about 300 μMto about 900 μM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 400 μM to about 800 μM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 500 μM to about 700 mM of the anti-fibrinolytic.In some embodiments, the solution includes a concentration of about 600μM. In some embodiments, the solution includes a concentration of about0.1 mM to about 1.0 M of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 1.0 mM to about 900 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 10 mM to about 800 mM of the anti-fibrinolytic.In some embodiments, the solution includes a concentration of about 50mM to about 700 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 100 mM to about 600 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 150 mM to about 500 mM of the anti-fibrinolytic.In some embodiments, the solution includes a concentration of about 200mM to about 400 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 250 mM to about 300 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 0.2 mM to about 9 mM of the anti-fibrinolytic. Insome embodiments, the solution includes a concentration of about 0.3 mMto about 8 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 0.4 mM to about 7 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 0.5 mM to about 6 mM of the anti-fibrinolytic. Insome embodiments, the solution includes a concentration of about 0.6 mMto about 5 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 0.7 mM to about 4 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 0.8 mM to about 3 mM of the anti-fibrinolytic. Insome embodiments, the solution includes a concentration of about 0.9 mMto about 2 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 1 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 10 mM to about 150 mM of the anti-fibrinolytic.In some embodiments, the solution includes a concentration of about 20mM to about 125 mM of the anti-fibrinolytic. In some embodiments, thesolution includes a concentration of about 30 mM to 100 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 40 mM to about 90 mM of the anti-fibrinolytic. Insome embodiments, the solution includes a concentration of about 50 mMto 80 mM of the anti-fibrinolytic. In some embodiments, the solutionincludes a concentration of about 60 mM to 70 mM of theanti-fibrinolytic. In some embodiments, the solution includes aconcentration of about 50 mM of the anti-fibrinolytic. In someembodiments, the solution includes a concentration of about 100 mM ofthe anti-fibrinolytic.

In some embodiments, the incubation of the anti-fibrinolytic in thepresence of the loading buffer is performed from about 1 minute to about4 hours. In some embodiments, the incubation is performed at anincubation period of from about 30 minutes to about 3 hours. In someembodiments, the incubation is performed at an incubation period of fromabout 1 hour to about 2 hours. In some embodiments, the incubation isperformed at an incubation period of about 3 hours.

In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 1 μM to about 100 mM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 10 μM to about 100 mM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 100 μM to about 10 mM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 200 μM to about 1 mM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 300 μM to about 900 μM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 400 μm to about 800 μM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is from about 500 μm to about 700 μM.In some embodiments, the concentration of anti-fibrinolytic in theanti-fibrinolytic loaded platelets is about 600 μM. In some embodiments,the concentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 0.1 mM to about 100 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 1.0 mM to about 900 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 10 mM to about 800 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 50 mM to about 700 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 100 mM to about 600 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 150 mM to about 500 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 200 mM to about 400 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 250 mM to about 300 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 1 mM to 100 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 5 mM to about 95 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 10 mM to about 90 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 15 mM to about 85 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 20 mM to about 80 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 25 mM to about 75 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 30 mM to about 70 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 35 mM to about 65 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 40 mM to about 60 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 45 mM to about 55 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 10 mM to about 100 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 20 mM to 90 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 30 mM to about 80 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 40 mM to 70 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is from about 50 mM to 60 mM. In some embodiments, theconcentration of anti-fibrinolytic in the anti-fibrinolytic loadedplatelets is about 50 mM. In some embodiments, the therapeuticallyeffective amount can be any of the concentrations described herein.

In some embodiments, the methods further include drying theanti-fibrinolytic loaded platelets. In some embodiments, the drying stepincludes freeze-drying the anti-fibrinolytic loaded platelets. In someembodiments, the methods further include rehydrating theanti-fibrinolytic loaded platelets obtained from the drying step.

In some embodiments, anti-fibrinolytic loaded platelets are prepared byusing any of the variety of methods provided herein.

In some embodiments, rehydrated anti-fibrinolytic loaded platelets areprepared by any one method comprising rehydrating the anti-fibrinolyticloaded platelets provided herein.

The anti-fibrinolytic loaded platelets can be used, for example, intherapeutic applications as disclosed herein. As described herein,platelets can stop bleeding by aggregating at an injury site which canbe further alleviated by anti-fibrinolytic loaded platelets. In someembodiments, the anti-fibrinolytic loaded platelets can be used to treattraumatic bleeding events, such as a hemorrhage. Hemorrhage occurs whenblood escapes outside its containing vessel (e.g., artery, vein,capillary, etc.) In some embodiments, the anti-fibrinolytic loadedplatelets can be used to treat an external hemorrhage. In someembodiments, the anti-fibrinolytic loaded platelets can be used to treatan internal hemorrhage. In some embodiments, the anti-fibrinolyticloaded platelets can be used to treat an external and an internalhemorrhage. In some embodiments, the anti-fibrinolytic loaded plateletscan be used to treat a surgical hemorrhage. In some embodiments, theanti-fibrinolytic loaded platelets can be used to treat a non-surgicalhemorrhage. In some embodiments, the anti-fibrinolytic loaded plateletscan be used to treat a grade (e.g., category) 1 hemorrhage. For example,a grade 1 hemorrhage can include petechial bleeding. In someembodiments, the anti-fibrinolytic loaded platelets can be used to treata grade 2 hemorrhage. For example, a grade (e.g., category) 2 hemorrhagecan include mild blood loss (e.g., a clinically significant amount ofblood). In some embodiments, the anti-fibrinolytic loaded platelets canbe used to treat a grade 3 hemorrhage. For example, a grade (e.g.,category 3) hemorrhage can include gross blood loss and can requiretransfusion. In some embodiments, the anti-fibrinolytic loaded plateletscan be used to treat a grade 4 hemorrhage. For example, a grade (e.g.,category) 4 hemorrhage can include debilitating blood loss, retinalblood loss, and/or cerebral blood loss associated with a fatality.

Unloaded platelets can be used, for example, in therapeutic applicationsas disclosed herein. For example, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat adisease. In some embodiments, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treathemophilia. In some embodiments, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat acquiredhemophilia. For example, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat acquiredhemophilia. In some embodiments, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat acquiredhemophilia.

In some embodiments, treatment of a subject with platelets loaded withan anti-fibrinolytic compound provides a “r” time (time to clot) that isshorter than the “r” time for treatment of the subject with the sameamount of the free anti-fibrinolytic compound, that is, theanti-fibrinolytic compound that is not loaded into the platelets.

In some embodiments, treatment of a subject with thrombosomes loadedwith an anti-fibrinolytic compound provides a “r” time (time to clot)that is shorter than the “r” time for treatment of the subject with thesame amount of the free anti-fibrinolytic compound, that is, theanti-fibrinolytic compound that is not loaded into the thrombosomes.

The anti-fibrinolytic loaded platelets can be used in therapeuticapplications as disclosed herein. For example, the anti-fibrinolyticloaded platelets can be used to treat hemophilia. In some embodiments,the anti-fibrinolytic loaded platelets can be used to treat acquired(e.g., hemophilia with an inhibitor antibody) hemophilia. Hemophilia isa disease in which the ability of blood to clot is severely reduced.Hemophilia can cause a subject to severely bleed even from a slightinjury. Classic hemophilia generally results from the deficiency of oneor more clotting factors. The various types of hemophilia generallyresult from a deficiency in one more clotting factors, such as, in anon-limiting way, Factor VII, Factor VIII, and Factor IX. Other types ofclotting factor deficiencies can also result in hemophilia, for example,clotting Factor XI. Some non-limiting examples of acquired hemophiliatypes include, for example, hemophilia A, hemophilia B, and hemophiliaC.

Hemophilia A and hemophilia B diseases are congenital X-linkedcoagulation disorders caused by the lack of or a defect in the generequired to produce active Factor VIII or Factor IX protein,respectively. Hemophilia often manifests in patients experiencingfrequent nosebleeds, easy bruising, and excessive bleeding duringmenstruation or invasive procedures. The disease affects about 20,000people in the United States. There are also acquired forms of hemophiliathat are caused by or exacerbated by neutralizing antibodies to FactorVIII or Factor IX. Factor VIII and Factor IX are produced in the liver.Plasma levels in a healthy human subject (“normal” levels) of FactorVIII are about 0.22 μg/mL and about 5 μg/mL for Factor IX. Factor VIIIand Factor IX are coagulation factors of the intrinsic pathway to repairdamaged surface of blood vessels. Additionally, thrombosomes potentiateclot formation and through alterative mechanisms reduce the timenecessary to clot formation when the clotting process is reduced by thelack of Factor VIII or Factor IX.

In some embodiments, platelets (e.g., anti-fibrinolytic loadedplatelets), lyophilized platelets (e.g., anti-fibrinolytic loadedlyophilized platelets) or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives) are used to treat a coagulopathy. In someembodiments, the coagulopathy is a drug-induced coagulopathy. In someembodiments, the coagulopathy occurs following administration of anantiplatelet agent. In some embodiments, the coagulopathy occursfollowing administration of an anticoagulant.

In some embodiments, a composition comprising platelets such aslyophilized platelets or platelet derivatives (any of which may beloaded to with anti-fibrinolytics) may be delivered to a wound on thesurface of or in the interior of a patient. In some embodiments, acomposition comprising platelets, lyophilized platelets, or plateletderivatives (any of which may be loaded to with anti-fibrinolytics) canbe applied in selected forms including, but not limited to, adhesivebandages, compression bandages, liquid solutions, aerosols, matrixcompositions, and coated sutures or other medical closures. In someembodiments, a platelet derivative (e.g. an anti-fibrinolytic loadedplatelet derivative) may be administered to all or only a portion of anaffected area on the surface of a patient. In other embodiments, acomposition comprising platelets such lyophilized platelets or plateletderivatives (any of which may be loaded to with anti-fibrinolytics) maybe administered systemically, for example via the blood stream. In someembodiments, an application of the platelet derivative (e.g. ananti-fibrinolytic loaded platelet derivative) can produce hemostaticeffects for 2 or 3 days, preferably 5 to 10 days, or most preferably forup to 14 days.

Some embodiments provide a method of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition comprisingplatelets such as lyophilized platelets (e.g. anti-fibrinolytic loadedlyophilized platelets) or platelet derivatives (e.g. anti-fibrinolyticloaded platelet derivatives) and a loading buffer comprising a salt, abase, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition prepared bya process comprising contacting platelets (e.g., anti-fibrinolyticloaded platelets) with a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent, to form thecomposition.

In some embodiments of any of the methods described herein, thecoagulopathy is the result of an anticoagulant.

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject is a subject need thereof, wherein the subject hasbeen treated or is being treated with an anticoagulant, the methodcomprising administering to the subject in need thereof atherapeutically effective amount of a composition comprising platelets(e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets(e.g., anti-fibrinolytic loaded lyophilized platelets) or plateletderivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and aloading buffer comprising a salt, a base, a loading agent, andoptionally at least one organic solvent.

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject is a subject need thereof, wherein the subject hasbeen treated or is being treated with an anticoagulant, the methodcomprising administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processcomprising contacting platelets (e.g., anti-fibrinolytic loadedplatelets) with a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent, to form thecomposition.

Some embodiments provide a method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition comprisingplatelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilizedplatelets (e.g., anti-fibrinolytic loaded lyophilized platelets) orplatelet derivatives (e.g., anti-fibrinolytic loaded plateletderivatives) and a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition prepared bya process comprising contacting platelets (e.g., anti-fibrinolyticloaded platelets) with a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent, to form thecomposition.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has been treated or is being treated withan anticoagulant, the method comprising administering to the subject inneed thereof a therapeutically effective amount of a compositioncomprising platelets (e.g., anti-fibrinolytic loaded platelets) such aslyophilized platelets (e.g., anti-fibrinolytic loaded lyophilizedplatelets) or anti-platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) and a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has been treated or is being treated withan anticoagulant, the method comprising administering to the subject inneed thereof a therapeutically effective amount of a compositionprepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition.

Compositions as described herein can also be administered to prepare asubject for surgery, in some cases. For some patients taking ananticoagulant, it may be difficult or impossible to reduce the dosage ofthe anticoagulant before surgery (e.g., in the case of trauma or otheremergency surgery). For some patients taking an anticoagulant, it may beinadvisable to reduce the dosage of the anticoagulant before surgery(e.g., if the patient would be at risk of a thrombotic event (e.g., deepvein thrombosis, pulmonary embolism, or stroke) if the dosage of theanticoagulant were reduced over time.

Accordingly, some embodiments provide a method of preparing a subjectfor surgery, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition comprisingplatelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilizedplatelets (e.g., anti-fibrinolytic loaded lyophilized platelets) orplatelet derivatives (e.g., anti-fibrinolytic loaded plateletderivatives) and a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery,the method comprising administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processcomprising contacting platelets (e.g., anti-fibrinolytic loadedplatelets) with a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent, to form thecomposition.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with ananticoagulant, the method comprising administering to the subject inneed thereof a therapeutically effective amount of a compositioncomprising platelets (e.g., anti-fibrinolytic loaded platelets) such aslyophilized platelets (e.g., anti-fibrinolytic loaded lyophilizedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) and a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with ananticoagulant, the method comprising administering to the subject inneed thereof a therapeutically effective amount of a compositionprepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition.

In some embodiments, a surgery can be an emergency surgery (e.g., in thecase of trauma) or a scheduled surgery.

In some embodiments of any of the methods described herein, treatmentwith an anticoagulant can be stopped (e.g., in preparation for surgery).In some embodiments, treatment with an anticoagulant can continue.

In some embodiments of any of the methods described herein, the subjectmay or may not be also treated with an anticoagulant reversal agent(e.g., idarucizumab, Andexanet Alfa, Ciraparantag (aripazine), protaminesulfate, vitamin K). In some embodiments, the subject is not alsotreated with an anticoagulant reversal agent. In some embodiments, thesubject is also treated with an anticoagulant reversal agent. It will beunderstood that an anticoagulant reversal agent can be chosen based onthe anticoagulant administered to the subject.

Some embodiments provide a method of ameliorating the effects of ananticoagulant in a subject, the method comprising administering to thesubject in need thereof a therapeutically effective amount of acomposition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) such as lyophilized platelets (e.g., anti-fibrinolytic loadedlyophilized platelets) or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives) and a loading buffer comprising a salt, abase, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of ameliorating the effects of ananticoagulant in a subject, the method comprising administering to thesubject in need thereof a therapeutically effective amount of acomposition prepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition.

In some embodiments, the effects of an anticoagulant may need to beameliorated due to an incorrect dosage of an anticoagulant. For example,in some embodiments, the effects of an anticoagulant can be amelioratedfollowing an overdose of the anticoagulant. In some cases, the effectsof an anticoagulant may need to be ameliorated due to a potential forinteraction with another drug (e.g., a second anticoagulant). Forexample, in some embodiments, the effects of an anticoagulant can beameliorated following an erroneous dosing of two or more drugs, at leastone of which is an anticoagulant. In some cases, the composition isadministered following administration to the subject or assumption bysubject, or an overdose of the anticoagulant.

In some embodiments, the anticoagulant is selected from the groupconsisting of an anti-factor IIa agent such as dabigatran (e.g.,PRADAXA®), argatroban, or hirudin; an anti-factor Xa agent such asrivaroxaban (e.g., XARELTO®), apixaban (e.g., ELIQUIS®), edoxaban (e.g.,SAVAYSA®), or fondaparinux (e.g., ARIXTRA®); a traditional anticoagulantsuch as warfarin (e.g., COUMADIN®) and heparin/LMWH (low molecularweight heparins); supplements such as herbal supplements, and acombination thereof. Examples of supplements include garlic, coenzymeCoQ10, glucosamine, glucosamine-condroitin sulfate. A non-limitingexample of an herbal supplement is garlic.

In some embodiments, the anticoagulant is dabigatran (e.g., PRADAXA®).

In some embodiments, the anticoagulant is argatroban.

In some embodiments, the anticoagulant is hirudin.

In some embodiments, the anticoagulant is rivaroxaban (e.g., XARELTO®).

In some embodiments, the anticoagulant is apixaban (e.g., ELIQUIS®).

In some embodiments, the anticoagulant is edoxaban (e.g., SAVAYSA®).

In some embodiments, the anticoagulant is fondaparinux (e.g., ARIXTRA®).

In some embodiments, the anticoagulant is heparin or a low molecularweight heparin (LMWH).

In some embodiments, the anticoagulant is warfarin (e.g., COUMADIN®).

In some embodiments, the anticoagulant is tifacogin.

In some embodiments, the anticoagulant is Factor VIIai.

In some embodiments, the anticoagulant is SB249417.

In some embodiments, the anticoagulant is pegnivacogin (with or withoutanivamersen).

In some embodiments, the anticoagulant is TTP889.

In some embodiments, the anticoagulant is idraparinux.

In some embodiments, the anticoagulant is idrabiotaparinux.

In some embodiments, the anticoagulant is SR23781A.

In some embodiments, the anticoagulant is apixaban.

In some embodiments, the anticoagulant is betrixaban.

In some embodiments, the anticoagulant is lepirudin.

In some embodiments, the anticoagulant is bivalirudin.

In some embodiments, the anticoagulant is ximelagatran.

In some embodiments, the anticoagulant is phenprocoumon.

In some embodiments, the anticoagulant is acenocoumarol.

In some embodiments, the anticoagulant an indandione.

In some embodiments, the anticoagulant is fluindione.

In some embodiments, the anticoagulant is a supplement.

In some embodiments, the anticoagulant is an herbal supplement.

In some embodiments, rehydrating the composition (e.g., any of thecompositions described herein) comprising platelets (e.g.,anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g.,anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives(e.g., anti-fibrinolytic loaded platelet derivatives) comprises addingto the platelets an aqueous liquid. In some embodiments, the aqueousliquid is water. In some embodiments, the aqueous liquid is an aqueoussolution (e.g., a buffer). In some embodiments, the aqueous liquid is asaline solution. In some embodiments, the aqueous liquid is asuspension.

In some embodiments, the rehydrated platelets (e.g., anti-fibrinolyticloaded platelets) or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives) have coagulation factor levels showing allindividual factors (e.g., Factors VII, VIII and IX) associated withblood clotting at 40 international units (IU) or greater.

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject is a subject need thereof, wherein the subject hasbeen treated or is being treated with an antiplatelet agent, the methodcomprising administering to the subject in need thereof atherapeutically effective amount of platelets (e.g., anti-fibrinolyticloaded platelets), lyophilized platelets (e.g., anti-fibrinolytic loadedlyophilized platelets), or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives).

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof a therapeutically effective amount of a compositionprepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has or has been treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof a therapeutically effective amount of a compositioncomprising platelets (e.g., anti-fibrinolytic loaded platelets) such aslyophilized platelets (e.g., anti-fibrinolytic loaded lyophilizedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) and a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition prepared bya process comprising contacting platelets (e.g., anti-fibrinolyticloaded platelets) with a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent, to form thecomposition.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has been treated or is being treated withan antiplatelet agent, the method comprising administering to thesubject in need thereof a therapeutically effective amount of acomposition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) such as lyophilized platelets (e.g., anti-fibrinolytic loadedlyophilized platelets) or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives) and loading buffer comprising a salt, abase, a loading agent, and optionally at least one organic solvent, toform the composition. Some embodiments provide a method of restoringnormal hemostasis in a subject, wherein the subject has been treated oris being treated with an antiplatelet agent, the method comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a composition prepared by a process comprising contactingplatelets (e.g., anti-fibrinolytic loaded platelets) with a loadingbuffer comprising a salt, a base, a loading agent, and optionally atleast one organic solvent.

Compositions as described herein can also be administered to prepare asubject for surgery, in some cases. For some patients taking anantiplatelet agent, it may be difficult or impossible to reduce thedosage of the antiplatelet agent before surgery (e.g., in the case oftrauma or other emergency surgery). For some patients taking anantiplatelet agent, it may be inadvisable to reduce the dosage of theantiplatelet agent before surgery (e.g., if the patient would be at riskof a thrombotic event (e.g., deep vein thrombosis, pulmonary embolism,or stroke) if the dosage of the antiplatelet agent were reduced overtime.

Accordingly, some embodiments provide a method of preparing a subjectfor surgery, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition comprisingplatelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilizedplatelets (e.g., anti-fibrinolytic loaded lyophilized platelets) orplatelet derivatives (e.g., anti-fibrinolytic loaded plateletderivatives) and a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery,the method comprising administering to the subject in need thereof atherapeutically effective amount of a composition prepared by a processcomprising contacting platelets (e.g., anti-fibrinolytic loadedplatelets) with a loading buffer comprising a salt, a base, a loadingagent, and optionally at least one organic solvent, to form thecomposition.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof a therapeutically effective amount of a compositioncomprising platelets (e.g., anti-fibrinolytic loaded platelets) such aslyophilized platelets (e.g., anti-fibrinolytic loaded lyophilizedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) and a loading buffer comprising a salt, a base, aloading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof a therapeutically effective amount of a compositionprepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition.

In some embodiments, a surgery can be an emergency surgery (e.g., in thecase of trauma) or a scheduled surgery.

In some embodiments, treatment with an anticoagulant can be stopped(e.g., in preparation for surgery). In some embodiments, treatment withan anticoagulant can continue.

Some embodiments provide a method of ameliorating the effects of anantiplatelet agent in a subject, the method comprising administering tothe subject in need thereof a therapeutically effective amount of acomposition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) such as lyophilized platelets (e.g., anti-fibrinolytic loadedlyophilized platelets) or platelet derivatives (e.g., anti-fibrinolyticloaded platelet derivatives) and contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent.

Some embodiments provide a method of ameliorating the effects of anantiplatelet agent in a subject, the method comprising administering tothe subject in need thereof a therapeutically effective amount of acomposition prepared by a process comprising contacting platelets (e.g.,anti-fibrinolytic loaded platelets) with a loading buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent, to form the composition.

In some cases, the effects of an antiplatelet agent may need to beameliorated due to an incorrect dosage of an antiplatelet agent. Forexample, in some embodiments, the effects of an antiplatelet agent canbe ameliorated following an overdose of the antiplatelet agent. In somecases, the effects of an antiplatelet agent may need to be ameliorateddue to a potential for interaction with another drug (e.g., a secondantiplatelet agent). For example, in some embodiments, the effects of anantiplatelet agent can be ameliorated following an erroneous dosing oftwo or more drugs, at least one of which is an antiplatelet agent. Insome cases, the composition is administered following administration tothe subject or assumption by subject, or an overdose of the antiplateletagent.

In some embodiments, the antiplatelet agent is selected from the groupconsisting of aspirin (also called acetylsalicylic acid or ASA); a P2Y12inhibitor such as cangrelor (e.g., KENGREAL®), ticagrelor (e.g.,BRILINTA®), clopidogrel (e.g., PLAVIX®), or prasugrel (e.g., EFFIENT®);a glycoprotein IIb/IIIa inhibitor such as eptifibatide (e.g.,INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g.,REOPRO®)); supplements such as herbal supplements; or a combination ofany thereof. Examples of supplements include ginger, ginseng, ginkgo,green tea, kava, saw palmetto, boldo (Peumus boldus), Danshen (Salviamiltiorrhiza), Dong quai (Angelica sinensis) papaya (Carica papaya),fish oil, and vitamin E. Examples of herbal supplements include ginger,ginseng, and ginkgo.

In some embodiments, the antiplatelet agent is aspirin.

In some embodiments, the antiplatelet agent is cangrelor (e.g.,KENGREAL®).

In some embodiments, the antiplatelet agent is ticagrelor (e.g.,BRILINTA®).

In some embodiments, the antiplatelet agent is clopidogrel (e.g.,PLAVIX®).

In some embodiments, the antiplatelet agent is prasugrel (e.g.,EFFIENT®).

In some embodiments, the antiplatelet agent is eptifibatide (e.g.,INTEGRILIN®).

In some embodiments, the antiplatelet agent is tirofiban (e.g.,AGGRASTAT®).

In some embodiments, the antiplatelet agent is abciximab (e.g.,REOPRO®).

In some embodiments, the antiplatelet agent is terutroban.

In some embodiments, the antiplatelet agent is picotamide.

In some embodiments, the antiplatelet agent is elinogrel.

In some embodiments, the antiplatelet agent is ticlopidine.

In some embodiments, the antiplatelet agent is ibuprofen.

In some embodiments, the antiplatelet agent is vorapaxar.

In some embodiments, the antiplatelet agent is atopaxar.

In some embodiments, the antiplatelet agent is cilostazol.

In some embodiments, the antiplatelet agent is prostaglandin E1.

In some embodiments, the antiplatelet agent is epoprostenol.

In some embodiments, the antiplatelet agent is dipyridamole.

In some embodiments, the antiplatelet agent is treprostinil sodium.

In some embodiments, the antiplatelet agent is sarpogrelate.

In some embodiments, the antiplatelet agent is a supplement.

In some embodiments, the antiplatelet agent is an herbal supplement.

Clotting parameters of blood (e.g., the subject's blood) can be assessedat any appropriate time during the methods described herein. Forexample, one or more clotting parameters of blood can be assessed beforeadministration of a composition comprising platelets (e.g.,anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g.,anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives(e.g., anti-fibrinolytic loaded platelet derivatives) as describedherein, e.g., in order to determine the need for administration of acomposition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) as described herein. As another example, one ormore clotting parameters of blood can be assessed after administrationof a composition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) as described herein, e.g., in order to determinethe effectiveness of the administered composition, to determine whetheradditional administration of the composition is warranted, or todetermine whether it is safe to perform a surgical procedure.

Accordingly, any of the methods described herein can include steps ofassessing one or more clotting parameters of blood before administrationof a composition comprising platelets (e.g., anti-fibrinolytic loadedplatelets) or platelet derivatives (e.g., anti-fibrinolytic loadedplatelet derivatives) as described herein, assessing one or moreclotting parameters of blood after administration of a compositioncomprising platelets (e.g., anti-fibrinolytic loaded platelets) orplatelet derivatives (e.g., anti-fibrinolytic loaded plateletderivatives) as described herein, or both.

Any appropriate method can be used to assess clotting parameters ofblood. Non-limiting examples of methods include the prothrombin timeassay, international normalized ratio (INR), thrombin generation (TGA;which can be used to generate parameters such as, e.g., peak thrombin,endogenous thrombin potential (ETP), and lag time), thromboeleastography(TEG), activated clotting time (ACT), and partial thromboplastin time(PTT or aPTT).

INR is a standard method of determining dosing, see equation below,where “PT(x)” is the result of the prothrombin time assay, while the ISIconstant is dependent on the manufacturer of the Tissue Factor used inthe prothrombin time assay.

INR=((PT(patient))/(PT(normal))){circumflex over ( )}(ISI constant)

Warfarin inhibits the synthesis of four major plasma proteins that areintegral to healthy clot formation. A therapeutic maintenance dose ofwarfarin is typically targeted to an INR of about 2.0 to about 3.0.Thrombosomes present a unique treatment to restore hemostasis in thepresence of warfarin-type drugs. Warfarin dose can be expressed by INR,a ratio that increases with the amount of warfarin (1 is a normalvalue).

In some embodiments, a subject has an INR of more than 2.0 (e.g., atleast 2.2, at least 2.4, at least 2.5, at least 2.6, at least 2.8, atleast 3.0, at least 3.2, at least 3.4, at least 3.5, at least 3.6, atleast 3.8, at least 4.0, at least 4.2, at least 4.4, at least 4.5, atleast 4.6, at least 4.8, or at least 5.0) before administration of acomposition comprising platelets such as lyophilized platelets orplatelet derivatives as described herein. In some embodiments, a subject(e.g., a subject being treated with an anticoagulant, such as warfarin)has an INR of from 2.0 to 3.0, such as from 2.2 to 2.8, such as from 2.4to 2.6, such as 2.5.

In some embodiments, a subject has a lower INR (or a normal INR) afteradministration of a composition comprising platelets such as lyophilizedplatelets or platelet derivatives as described herein. For example, asubject can have an INR of 3.0 or less (e.g., less than 2.8, less than2.6, less than 2.5, less than 2.4, less than 2.2, less than 2.0, lessthan 1.8, less than 1.6, less than 1.5, less than 1.4, less than 1.2, orless than 1.0) after administration of a composition comprisingplatelets or platelet derivatives ad described herein.

Additionally or alternatively, the anti-fibrinolytic loaded plateletscan be employed in functional assays. In some embodiments, theanti-fibrinolytic loaded platelets are cold stored, cryopreserved, orlyophilized (to produce thrombosomes) prior to use in therapy or infunctional assays.

Any known technique for drying platelets can be used in accordance withthe present disclosure, as long as the technique can achieve a finalresidual moisture content of less than 5%. Preferably, the techniqueachieves a final residual moisture content of less than 2%, such as 1%,0.5%, or 0.1%. Non-limiting examples of suitable techniques arefreeze-drying (lyophilization) and spray-drying. A suitablelyophilization method is presented in Table A. Additional exemplarylyophilization methods can be found in U.S. Pat. Nos. 7,811,558,8,486,617, and 8,097,403, each of which are incorporated herein byreference in their entireties. An exemplary spray-drying methodincludes: combining nitrogen, as a drying gas, with a loading bufferaccording to the present disclosure, then introducing the mixture intoGEA Mobile Minor spray dryer from GEA Processing Engineering, Inc.(Columbia Md., USA), which has a Two-Fluid Nozzle configuration, spraydrying the mixture at an inlet temperature in the range of 150° C. to190° C., an outlet temperature in the range of 65° C. to 100° C., anatomic rate in the range of 0.5 to 2.0 bars, an atomic rate in the rangeof 5 to 13 kg/hr, a nitrogen use in the range of 60 to 100 kg/hr, and arun time of 10 to 35 minutes. The final step in spray drying ispreferentially collecting the dried mixture. The dried composition insome embodiments is stable for at least six months at temperatures thatrange from −20° C. or lower to 90° C. or higher.

TABLE A Exemplary Lyophilization Protocol Step Temp. Set Type DurationPressure Set Freezing Step F1 −50° C. Ramp Var N/A F2 −50° C. Hold 3 HrsN/A Vacuum Pulldown F3 −50° C. Hold Var N/A Primary Dry P1 −40° Hold 1.5Hrs 0 mT P2 −35° Ramp 2 Hrs 0 mT P3 −25° Ramp 2 Hrs 0 mT P4 −17° C. Ramp2 Hrs 0 mT P5  0° C. Ramp 1.5 Hrs 0 mT P6  27° C. Ramp 1.5 Hrs 0 mT P7 27° C. Hold 16 Hrs 0 mT Second Dry S1  27° C. Hold >8 Hrs 0 mT

In some embodiments, the step of drying the anti-fibrinolytic loadedplatelets that are obtained as disclosed herein, such as the step offreeze-drying the anti-fibrinolytic loaded platelets that are obtainedas disclosed herein, includes incubating the platelets with alyophilizing agent. In some embodiments, the lyophilizing agent ispolysucrose. In some embodiments, the lyophilizing agent is anon-reducing disaccharide. Accordingly, in some embodiments, the methodsfor preparing anti-fibrinolytic loaded platelets further includeincubating the anti-fibrinolytic loaded platelets with a lyophilizingagent. In some embodiments, the lyophilizing agent is a saccharide. Insome embodiments, the saccharide is a disaccharide, such as anon-reducing disaccharide.

In some embodiments, the step of drying the platelets that are obtainedas disclosed herein, such as the step of freeze-drying the plateletsthat are obtained as disclosed herein, includes incubating the plateletswith a lyophilizing agent to generate thrombosomes. In some embodiments,the lyophilizing agent is polysucrose. In some embodiments, thelyophilizing agent is a non-reducing disaccharide. Accordingly, in someembodiments, the methods for preparing thrombosomes from plateletsfurther include incubating the platelets with a lyophilizing agent. Insome embodiments, the lyophilizing agent is a saccharide. In someembodiments, the saccharide is a disaccharide, such as a non-reducingdisaccharide.

In some embodiments, the platelets are incubated with a lyophilizingagent for a sufficient amount of time and at a suitable temperature toload the platelets with the lyophilizing agent. Non-limiting examples ofsuitable lyophilizing agents are saccharides, such as monosaccharidesand disaccharides, including sucrose, maltose, trehalose, glucose (e.g.,dextrose), mannose, and xylose. In some embodiments, non-limitingexamples of lyophilizing agent include serum albumin, dextran, polyvinylpyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In someembodiments, exemplary lyophilizing agents can include a high molecularweight polymer, into the loading composition. By “high molecular weight”it is meant a polymer having an average molecular weight of about orabove 70 kDa. Non-limiting examples are polymers of sucrose andepichlorohydrin. In some embodiments, the lyophilizing agent ispolysucrose. Although any amount of high molecular weight polymer can beused as a lyophilizing agent, it is preferred that an amount be usedthat achieves a final concentration of about 3% to 10% (w/v), such as 3%to 7%, for example 6%.

In some embodiments, the process for preparing a composition includesadding an organic solvent, such as ethanol, to the loading solution. Insuch a loading solution, the solvent can range from 0.1% to 5.0% (v/v).

Within the process provided herein for making the compositions providedherein, addition of the lyophilizing agent can be the last step prior todrying. However, in some embodiments, the lyophilizing agent is added atthe same time or before the anti-fibrinolytic, the cryoprotectant, orother components of the loading composition. In some embodiments, thelyophilizing agent is added to the loading solution, thoroughly mixed toform a drying solution, dispensed into a drying vessel (e.g., a glass orplastic serum vial, a lyophilization bag), and subjected to conditionsthat allow for drying of the solution to form a dried composition.

An exemplary saccharide for use in the compositions disclosed herein istrehalose. Regardless of the identity of the saccharide, it can bepresent in the composition in any suitable amount. For example, it canbe present in an amount of 1 mM to 1 M. In embodiments, it is present inan amount of from 10 mM 10 to 500 mM. In some embodiments, it is presentin an amount of from 20 mM to 200 mM. In some embodiments, it is presentin an amount from 40 mM to 100 mM. In various embodiments, thesaccharide is present in different specific concentrations within theranges recited above, and one of skill in the art can immediatelyunderstand the various concentrations without the need to specificallyrecite each herein. Where more than one saccharide is present in thecomposition, each saccharide can be present in an amount according tothe ranges and particular concentrations recited above.

The step of incubating the platelets to load them with a cryoprotectantor as a lyophilizing agent includes incubating the platelets for a timesuitable for loading, as long as the time, taken in conjunction with thetemperature, is sufficient for the cryoprotectant or lyophilizing agentto come into contact with the platelets and, preferably, beincorporated, at least to some extent, into the platelets. Inembodiments, incubation is carried out for about 1 minute to about 180minutes or longer.

The step of incubating the platelets to load them with a cryoprotectantor lyophilizing agent includes incubating the platelets and thecryoprotectant at a temperature that, when selected in conjunction withthe amount of time allotted for loading, is suitable for loading. Ingeneral, the composition is incubated at a temperature above freezingfor at least a sufficient time for the cryoprotectant or lyophilizingagent to come into contact with the platelets. In embodiments,incubation is conducted at 37° C. In certain embodiments, incubation isperformed at 20° C. to 42° C. For example, in embodiments, incubation isperformed at 35° C. to 40° C. (e.g., 37° C.) for 110 to 130 (e.g., 120)minutes.

In various embodiments, the bag is a gas-permeable bag configured toallow gases to pass through at least a portion or all portions of thebag during the processing. The gas-permeable bag can allow for theexchange of gas within the interior of the bag with atmospheric gaspresent in the surrounding environment. The gas-permeable bag can bepermeable to gases, such as oxygen, nitrogen, water, air, hydrogen, andcarbon dioxide, allowing gas exchange to occur in the compositionsprovided herein. In some embodiments, the gas-permeable bag allows forthe removal of some of the carbon dioxide present within an interior ofthe bag by allowing the carbon dioxide to permeate through its wall. Insome embodiments, the release of carbon dioxide from the bag can beadvantageous to maintaining a desired pH level of the compositioncontained within the bag.

In some embodiments, the container of the process herein is agas-permeable container that is closed or sealed. In some embodiments,the container is a container that is closed or sealed and a portion ofwhich is gas-permeable. In some embodiments, the surface area of agas-permeable portion of a closed or sealed container (e.g., bag)relative to the volume of the product being contained in the container(hereinafter referred to as the “SA/V ratio”) can be adjusted to improvepH maintenance of the compositions provided herein. For example, in someembodiments, the SA/V ratio of the container can be at least about 2.0cm²/mL (e.g., at least about 2.1 cm²/mL, at least about 2.2 cm²/mL, atleast about 2.3 cm²/mL, at least about 2.4 cm²/mL, at least about 2.5cm²/mL, at least about 2.6 cm²/mL, at least about 2.7 cm²/mL, at leastabout 2.8 cm²/mL, at least about 2.9 cm²/mL, at least about 3.0 cm²/mL,at least about 3.1 cm²/mL, at least about 3.2 cm²/mL, at least about 3.3cm²/mL, at least about 3.4 cm²/mL, at least about 3.5 cm²/mL, at leastabout 3.6 cm²/mL, at least about 3.7 cm²/mL, at least about 3.8 cm²/mL,at least about 3.9 cm²/mL, at least about 4.0 cm²/mL, at least about 4.1cm²/mL, at least about 4.2 cm²/mL, at least about 4.3 cm²/mL, at leastabout 4.4 cm²/mL, at least about 4.5 cm²/mL, at least about 4.6 cm²/mL,at least about 4.7 cm²/mL, at least about 4.8 cm²/mL, at least about 4.9cm²/mL, or at least about 5.0 cm²/mL. In some embodiments, the SA/Vratio of the container can be at most about 10.0 cm²/mL (e.g., at mostabout 9.9 cm²/mL, at most about 9.8 cm²/mL, at most about 9.7 cm²/mL, atmost about 9.6 cm²/mL, at most about 9.5 cm²/mL, at most about 9.4cm²/mL, at most about 9.3 cm²/mL, at most about 9.2 cm²/mL, at mostabout 9.1 cm²/mL, at most about 9.0 cm²/mL, at most about 8.9 cm²/mL, atmost about 8.8 cm²/mL, at most about 8.7 cm²/mL, at most about 8.6,cm²/mL at most about 8.5 cm²/mL, at most about 8.4 cm²/mL, at most about8.3 cm²/mL, at most about 8.2 cm²/mL, at most about 8.1 cm²/mL, at mostabout 8.0 cm²/mL, at most about 7.9 cm²/mL, at most about 7.8 cm²/mL, atmost about 7.7 cm²/mL, at most about 7.6 cm²/mL, at most about 7.5cm²/mL, at most about 7.4 cm²/mL, at most about 7.3 cm²/mL, at mostabout 7.2 cm²/mL, at most about 7.1 cm²/mL, at most about 6.9 cm²/mL, atmost about 6.8 cm²/mL, at most about 6.7 cm²/mL, at most about 6.6cm²/mL, at most about 6.5 cm²/mL, at most about 6.4 cm²/mL, at mostabout 6.3 cm²/mL, at most about 6.2 cm²/mL, at most about 6.1 cm²/mL, atmost about 6.0 cm²/mL, at most about 5.9 cm²/mL, at most about 5.8cm²/mL, at most about 5.7 cm²/mL, at most about 5.6 cm²/mL, at mostabout 5.5 cm²/mL, at most about 5.4 cm²/mL, at most about 5.3 cm²/mL, atmost about 5.2 cm²/mL, at most about 5.1 cm²/mL, at most about 5.0cm²/mL, at most about 4.9 cm²/mL, at most about 4.8 cm²/mL, at mostabout 4.7 cm²/mL, at most about 4.6 cm²/mL, at most about 4.5 cm²/mL, atmost about 4.4 cm²/mL, at most about 4.3 cm²/mL, at most about 4.2cm²/mL, at most about 4.1 cm²/mL, or at most about 4.0 cm²/mL. In someembodiments, the SA/V ratio of the container can range from about 2.0 toabout 10.0 cm²/mL (e.g., from about 2.1 cm²/mL to about 9.9 cm²/mL, fromabout 2.2 cm²/mL to about 9.8 cm²/mL, from about 2.3 cm²/mL to about 9.7cm²/mL, from about 2.4 cm²/mL to about 9.6 cm²/mL, from about 2.5 cm²/mLto about 9.5 cm²/mL, from about 2.6 cm²/mL to about 9.4 cm²/mL, fromabout 2.7 cm²/mL to about 9.3 cm²/mL, from about 2.8 cm²/mL to about 9.2cm²/mL, from about 2.9 cm²/mL to about 9.1 cm²/mL, from about 3.0 cm²/mLto about 9.0 cm²/mL, from about 3.1 cm²/mL to about 8.9 cm²/mL, fromabout 3.2 cm²/mL to about 8.8 cm²/mL, from about 3.3 cm²/mL to about 8.7cm²/mL, from about 3.4 cm²/mL to about 8.6 cm²/mL, from about 3.5 cm²/mLto about 8.5 cm²/mL, from about 3.6 cm²/mL to about 8.4 cm²/mL, fromabout 3.7 cm²/mL to about 8.3 cm²/mL, from about 3.8 cm²/mL to about 8.2cm²/mL, from about 3.9 cm²/mL to about 8.1 cm²/mL, from about 4.0 cm²/mLto about 8.0 cm²/mL, from about 4.1 cm²/mL to about 7.9 cm²/mL, fromabout 4.2 cm²/mL to about 7.8 cm²/mL, from about 4.3 cm²/mL to about 7.7cm²/mL, from about 4.4 cm²/mL to about 7.6 cm²/mL, from about 4.5 cm²/mLto about 7.5 cm²/mL, from about 4.6 cm²/mL to about 7.4 cm²/mL, fromabout 4.7 cm²/mL to about 7.3 cm²/mL, from about 4.8 cm²/mL to about 7.2cm²/mL, from about 4.9 cm²/mL to about 7.1 cm²/mL, from about 5.0 cm²/mLto about 6.9 cm²/mL, from about 5.1 cm²/mL to about 6.8 cm²/mL, fromabout 5.2 cm²/mL to about 6.7 cm²/mL, from about 5.3 cm²/mL to about 6.6cm²/mL, from about 5.4 cm²/mL to about 6.5 cm²/mL, from about 5.5 cm²/mLto about 6.4 cm²/mL, from about 5.6 cm²/mL to about 6.3 cm²/mL, fromabout 5.7 cm²/mL to about 6.2 cm²/mL, or from about 5.8 cm²/mL to about6.1 cm²/mL.

Gas-permeable closed containers (e.g., bags) or portions thereof can bemade of one or more various gas-permeable materials. In someembodiments, the gas-permeable bag can be made of one or more polymersincluding fluoropolymers (such as polytetrafluoroethylene (PTFE) andperfluoroalkoxy (PFA) polymers), polyolefins (such as low-densitypolyethylene (LDPE), high-density polyethylene (HDPE)), fluorinatedethylene propylene (FEP), polystyrene, polyvinylchloride (PVC),silicone, and any combinations thereof.

In some embodiments, the lyophilizing agent as disclosed herein may be ahigh molecular weight polymer. By “high molecular weight” it is meant apolymer having an average molecular weight of about or above 70 kDa andup to 1,000,000 kDa Non-limiting examples are polymers of sucrose andepichlorohydrin (polysucrose). Although any amount of high molecularweight polymer can be used, it is preferred that an amount be used thatachieves a final concentration of about 3% to 10% (w/v), such as 3% to7%, for example 6%. Other non-limiting examples of lyoprotectants areserum albumin, dextran, polyvinyl pyrrolidone (PVP), starch, andhydroxyethyl starch (HES). In some embodiments, a lyoprotectant is alsoa cryoprotectant. For example, albumin, polysucrose, and sucrose canalso be used as a cryoprotectant.

In some embodiments, lyophilized platelets can be fixed (e.g.,lyophilized fixed plates) in fixing agent. In some embodiments,lyophilized platelets can be fixed in formalin (e.g., lyophilizedformalin-fixed platelets).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) canbe at a concentration from about 1,000 k/μl to about 500,000 k/μl. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 5,000 k/μl to about 450,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 10,000 k/μl to about 400,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 30,000 k/μl to about 300,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 40,000 k/μl to about 250,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 50,000 k/μl to about 225,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 60,000 k/μl to about 200,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 70,000 k/μl to about 175,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 80,000 k/μl to about 150,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 90,000 k/μl to about 125,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 100,000 k/μl to about 120,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 105,000 k/μl to about 115,000 k/μl. In someembodiments, the therapeutically effective amount of lyophilizedplatelets (e.g., thrombosomes) can be at any of the concentrationsdescribed herein).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) canbe at a concentration from about 1×10² particles/kg to from about 1×10¹³particles/kg. In some embodiments, the lyophilized platelets (e.g.,thrombosomes) can be at a concentration from about 1×10³ particles/kg tofrom about 1×10¹² particles/kg. In some embodiments, the lyophilizedplatelets (e.g., thrombosomes) can be at a concentration from about1×10⁴ particles/kg to from about 1×10¹¹ particles/kg. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 1×10⁵ particles/kg to from about 1×10¹⁰particles/kg. In some embodiments, the lyophilized platelets (e.g.,thrombosomes) can be at a concentration from about 1×10⁶ particles/kg tofrom about 1×10⁹ particles/kg. In some embodiments, the lyophilizedplatelets (e.g., thrombosomes) can be at a concentration from about1×10⁷ particles/kg to from about 1×10⁸ particles/kg. In someembodiments, a therapeutically effective amount of the lyophilizedplatelets (e.g., thrombosomes) can be at any of the concentrationsdescribed herein.

In some embodiments of the methods herein, any of the compositionsdescribed herein are administered topically. In some embodiments,topical administration can include administration via a solution, cream,gel, suspension, putty, particulates, or powder. In some embodiments,topical administration can include administration via a bandage (e.g. anadhesive bandage or a compression bandage) or medical closure (e.g.,sutures, staples)); for example the anti-fibrinolytic loaded plateletderivatives (e.g., lyopreserved platelets (e.g., thrombosomes)) can beembedded therein or coated thereupon), as described in PCT PublicationNo. WO2017/040238 (e.g., paragraphs [013]-[069]), corresponding to U.S.patent application Ser. No. 15/776,255, the entirety of which is hereinincorporated by reference.

In some embodiments of the methods herein, the compositions describedherein are administered parenterally.

In some embodiments of the methods herein, the compositions describedherein are administered intravenously.

In some embodiments of the methods herein, the compositions describedherein are administered intramuscularly.

In some embodiments of the methods herein, the compositions describedherein are administered intrathecally.

In some embodiments of the methods herein, the compositions describedherein are administered subcutaneously.

In some embodiments of the methods herein, the compositions describedherein are administered intraperitoneally. In some embodiments, theanti-fibrinolytic loaded platelets prepared as disclosed herein have astorage stability that is at least about equal to that of the plateletsprior to the loading of the anti-fibrinolytic.

The loading buffer may be any buffer that is non-toxic to the plateletsand provides adequate buffering capacity to the solution at thetemperatures at which the solution will be exposed during the processprovided herein. Thus, the buffer may include any of the knownbiologically compatible buffers available commercially, such asphosphate buffers, such as phosphate buffered saline (PBS),bicarbonate/carbonic acid, such as sodium-bicarbonate buffer,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), andtris-based buffers, such as tris-buffered saline (TB S). Likewise, itmay include one or more of the following buffers:propane-1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic;maleic; 2,2-dimethyl succinic; 3,3-dimethylglutaric;bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS);benzenehexacarboxylic (mellitic); N-(2-acetamido)imino-diacetic acid(ADA); butane-1,2,3,4-tetracarboxylic; pyrophosphoric;1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid);piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES);N-(2-acetamido)-2-amnoethanesulfonic acid (ACES);1,1-cyclohexanediacetic; 3,6-endomethylene-1,2,3,6-tetrahydrophthalicacid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride(CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);2-methylpropane-1,2,3-triscarboxylic (beta-methyltricarballylic);2-(N-morpholino)propane-sulfonic acid (MOPS); phosphoric; andN-tris(hydroxymethyl)methyl-2-amminoethane sulfonic acid (TES).

A plate reader (e.g., Tecan Microplate reader (e.g., Infinite® 200 PRO))can be used to quantify loading efficiency of the anti-fibrinolytic inthe anti-fibrinolytic loaded platelets. Platelets can be evaluated forfunctionality by adenosine diphosphate (ADP), collagen, arachidonicacid, phorbol myristate acetate (PMA), thrombin receptor activatingpeptide (TRAP), and/or any other platelet agonist known in the art forstimulation post-loading. A hemostasis analyzer (e.g., TEG® 5000Thromboelastogram® Hemostasis Analyzer system) can be used to testanti-fibrinolytic function of EACA loaded platelets.

In some embodiments, the anti-fibrinolytic platelets are lyophilized. Insome embodiments, the anti-fibrinolytic loaded platelets arecryopreserved. In some embodiments, the unloaded platelets arelyophilized. In some embodiments, the unloaded platelets arecryopreserved.

In some embodiments, the anti-fibrinolytic loaded platelets retain theloaded anti-fibrinolytic compound upon rehydration and release theanti-fibrinolytic compound upon stimulation by endogenous plateletactivators, such as endogenous platelet activators described herein.

In some embodiments, the dried platelets (such as freeze-driedplatelets) retain the loaded anti-fibrinolytic upon rehydration andrelease the anti-fibrinolytic (e.g., EACA) upon stimulation byendogenous platelet activators. In some embodiments, at least about 10%,such as at least about 20%, such as at least about 30% of theanti-fibrinolytic is retained. In some embodiments, from about 10% toabout 20%, such as from about 20% to about 30% of the anti-fibrinolyticis retained.

In some embodiments, anti-fibrinolytic loaded platelets,anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolyticloaded thrombosomes can shield the anti-fibrinolytic from exposure incirculation, thereby reducing or eliminating systemic toxicity (e.g.cardiotoxicity) associated with the anti-fibrinolytic. In someembodiments, anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, and/or anti-fibrinolytic loadedthrombosomes can also protect the anti-fibrinolytic from metabolicdegradation or inactivation. In some embodiments, anti-fibrinolyticdelivery with anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, and/or anti-fibrinolytic loadedthrombosomes can therefore be advantageous in treatment of diseases suchas traumatic bleeding events (e.g., hemorrhage) and/or hemophilia, sinceanti-fibrinolytic loaded platelets, anti-fibrinolytic loaded plateletderivatives, and/or anti-fibrinolytic loaded thrombosomes can mitigatesystemic side effects. In some embodiments, anti-fibrinolytic loadedplatelets, anti-fibrinolytic loaded platelet derivatives, and/oranti-fibrinolytic loaded thrombosomes can be used in any therapeuticsetting in which expedited healing process is required or advantageous.

In some embodiments, provided herein is a method of treating a diseaseas disclosed herein in a subject in need thereof, comprisingadministering anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes asdisclosed herein. In some embodiments, provided herein is a method oftreating a disease as disclosed herein in a subject in need thereof,comprising administering cold stored, room temperature stored,cryopreserved, thawed, rehydrated, and/or lyophilized platelets,platelet derivatives, or thrombosomes as disclosed herein. In someembodiments, the disease is hemophilia B. In some embodiments, thedisease is acquired hemophilia B. In some embodiments the disease ishemophilia A. In some embodiments, the disease is acquired hemophilia A.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingcontacting platelets with an anti-fibrinolytic and with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingproviding platelets and contacting the platelets with ananti-fibrinolytic and with a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent, to form theanti-fibrinolytic loaded platelets. In some embodiments of preparinganti-fibrinolytic loaded platelets, the platelets are contacted with theanti-fibrinolytic and with the loading buffer sequentially, in eitherorder.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingcontacting platelets with the anti-fibrinolytic to form a firstcomposition and contacting the first composition with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingcontacting the platelets with a buffer including a salt, a base, aloading agent, and optionally at least one organic solvent to form afirst composition and contacting the first composition with ananti-fibrinolytic, to form the anti-fibrinolytic loaded platelets. Insome embodiments of preparing anti-fibrinolytic loaded platelets, theplatelets are contacted with the anti-fibrinolytic and with the loadingbuffer concurrently.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingcontacting the platelets with an anti-fibrinolytic in the presence of aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent to form the anti-fibrinolytic-loadedplatelets. In some embodiments of preparing anti-fibrinolytic loadedplatelets, the platelets are pooled from a plurality of donors prior toa treating step.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingA) pooling platelets from a plurality of donors and B) contacting theplatelets from step (A) with an anti-fibrinolytic and with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, to form the anti-fibrinolytic loadedplatelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingA) pooling platelets from a plurality of donors and B) contacting theplatelets from step (A) with an anti-fibrinolytic to form a firstcomposition and contacting the first composition with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingA) pooling platelets from a plurality of donors and B) contacting theplatelets from step (A) with an anti-fibrinolytic to form a firstcomposition and contacting the first composition with a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingA) pooling platelets from a plurality of donors and B) contacting theplatelets from step (A) with a loading buffer including a salt, a base,a loading agent, and optionally at least one organic solvent, to form afirst composition and contacting the first composition with ananti-fibrinolytic to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded platelets, wherein theanti-fibrinolytic loaded platelets are prepared by a process comprisingA) pooling platelets from a plurality of donors and B) contacting theplatelets with an anti-fibrinolytic in the presence of a loading bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising contacting thrombosomes with an anti-fibrinolytic and with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes prepared by aprocess comprising providing platelets and contacting the platelets withan anti-fibrinolytic and with a loading buffer including a salt, a base,a loading agent, and optionally at least one organic solvent, and a stepof freeze-drying, to form the anti-fibrinolytic loaded thrombosomes. Insome embodiments of preparing anti-fibrinolytic loaded platelets, theplatelets are contacted with the anti-fibrinolytic and with the loadingbuffer sequentially, in either order.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising contacting platelets with the anti-fibrinolytic to form afirst composition and contacting the first composition with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising contacting platelets with a buffer including a salt, a base,a loading agent, and optionally at least one organic solvent to form afirst composition and contacting the first composition with ananti-fibrinolytic, and a freeze drying step, to form theanti-fibrinolytic loaded thrombosomes. In some embodiments of preparinganti-fibrinolytic loaded thrombosomes, the platelets are contacted withthe anti-fibrinolytic and with the loading buffer concurrently.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising contacting platelets with an anti-fibrinolytic in thepresence of a loading buffer including a salt, a base, a loading agent,and optionally at least one organic solvent, and a step of freeze-dryingto form the anti-fibrinolytic-loaded thrombosomes. In some embodimentsof preparing anti-fibrinolytic loaded thrombosomes, the platelets arepooled from a plurality of donors prior to a treating step.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising A) pooling platelets from a plurality of donors and B)contacting the platelets from step (A) with an anti-fibrinolytic andwith a loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, and a freeze-drying step, toform the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising A) pooling platelets from a plurality of donors and B)contacting the platelets from step (A) with an anti-fibrinolytic to forma first composition and contacting the first composition with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising A) pooling platelets from a plurality of donors and B)contacting the platelets from step (A) with an anti-fibrinolytic to forma first composition and contacting the first composition with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising A) pooling platelets from a plurality of donors and B)contacting the platelets from step (A) with a loading buffer including asalt, a base, a loading agent, and optionally at least one organicsolvent, to form a first composition and contacting the firstcomposition with an anti-fibrinolytic, and a step of freeze-drying toform the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic loaded thrombosomes are prepared by a processcomprising A) pooling platelets from a plurality of donors and B)contacting the platelets with an anti-fibrinolytic in the presence of aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising contactingthrombosomes with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes prepared by a processcomprising providing platelets and contacting the platelets with aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, and a step of freeze-drying, to form theunloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising contacting plateletswith a loading buffer including a salt and a base to form a firstcomposition and contacting the first composition with a loading agent,and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising contacting plateletswith a loading agent, and optionally at least one organic solvent toform a first composition and contacting the first composition with aloading buffer including a salt and a base, and a freeze-drying step, toform the unloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising contacting platelets aloading buffer including a salt, a base, a loading agent, and optionallyat least one organic solvent, and a step of freeze-drying to form theanti-fibrinolytic-loaded thrombosomes. In some embodiments of preparingunloaded thrombosomes, the platelets are pooled from a plurality ofdonors prior to a treating step.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising A) pooling plateletsfrom a plurality of donors and B) contacting the platelets from step (A)with a loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, and a freeze-drying step, toform the unloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising A) pooling plateletsfrom a plurality of donors and B) contacting the platelets from step (A)with a loading buffer including a salt and a base to form a firstcomposition and contacting the first composition with a loading agent,and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising A) pooling plateletsfrom a plurality of donors and B) contacting the platelets from step (A)with an a loading agent to form a first composition and contacting thefirst composition with a loading buffer including a salt and a base, andoptionally at least one organic solvent, and a step of freeze-drying, toform the unloaded thrombosomes.

In some embodiments, no solvent is used. Thus, provided herein aremethods to treat acquired hemophilia (e.g., acquired hemophilia A oracquired hemophilia B), comprising a therapeutically effective amount ofanti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolyticthrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol, and    -   C) a step of freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with an anti-fibrinolytic to form a        first composition;    -   C) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the anti-fibrinolytic        loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol, and    -   (D) a step freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition;    -   C) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   D) a step of freeze drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) preparing platelets;    -   B) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets, wherein the        method does not comprise contacting the platelets with an        organic solvent such as ethanol, and    -   C) a step of freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   a) preparing platelets;    -   b) contacting the platelets with an anti-fibrinolytic to form a        first composition;    -   c) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the anti-fibrinolytic        loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   d) a step of freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat acquired hemophilia (e.g., acquiredhemophilia A or acquired hemophilia B), comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   a) preparing platelets;    -   b) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition;    -   c) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets.    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   d) a freeze-drying step, to form the anti-fibrinolytic loaded        thrombosomes.

In some embodiments, unloaded platelets, unloaded platelet derivatives,and/or unloaded thrombosomes can be advantageous in the treatment ofdiseases such as a hemophilia (e.g., acquired hemophilia). In someembodiments, unloaded platelets, unloaded platelet derivatives, and/orunloaded thrombosomes can be advantageous in the treatment of diseasesuch as acquired hemophilia (e.g., hemophilia with an inhibitorantibody).

In some embodiments, provided herein is a method of treating a diseaseas disclosed herein in a subject in need thereof, (e.g., acquiredhemophilia A, acquired hemophilia B), comprising administering to asubject in need thereof, unloaded platelets, unloaded plateletderivatives, or unloaded thrombosomes as disclosed herein. In someembodiments, provided herein is a method of treating a disease asdisclosed herein in a subject in need thereof, comprising administeringunloaded cold stored, room temperature stored, cryopreserved thawed,rehydrated, and/or lyophilized platelets, unloaded platelet derivatives,or unloaded thrombosomes as disclosed herein.

In some embodiments, provided herein are methods of treatment of adisease with anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes asdisclosed herein. For example, the treatment of a disease can beperformed in a model for a disease (e.g., Hemophilia A, Hemophilia B).For example, plasma chemically depleted of Factor VIII (e.g., plasmasubstantially similar to plasma from a subject with hemophilia A) can betreated with anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes asdisclosed herein. For example, plasma immuno-depleted of Factor IX(e.g., plasma substantially similar to plasma from a subject withhemophilia B) can be treated with anti-fibrinolytic loaded platelets,anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolyticloaded thrombosomes as disclosed herein.

In some embodiments, provided herein are methods of treatment of adisease including administering to a subject in need thereof, unloadedplatelets, unloaded platelet derivatives, or unloaded thrombosomes asdisclosed herein. For example, the treatment of a disease can beperformed in a model for a disease (e.g., Hemophilia A, Hemophilia B).For example, plasma chemically depleted of Factor VIII (e.g., plasmasubstantially similar to plasma from a subject with hemophilia A) can betreated with unloaded platelets, unloaded platelet derivatives, orunloaded thrombosomes. For example, plasma with antibodies to eitherFactor VIII or Factor IX (e.g., plasma substantially similar to plasmafrom a subject with hemophilia B) can be treated with unloadedplatelets, unloaded platelet derivatives, or unloaded thrombosomes.

In some embodiments, provided herein are methods of treatment of adisease as disclosed herein, including administering unloaded coldstored, room temperature stored, cryopreserved thawed, rehydrated,and/or lyophilized platelets, unloaded platelet derivatives, or unloadedthrombosomes as disclosed herein to a model of a disease (e.g.,Hemophilia A, Hemophilia B).

A loading agent (e.g., an incubating agent) can include any appropriatecomponents. In some embodiments, the loading agent may comprise a liquidmedium. In some embodiments the loading agent may comprise one or moresalts selected from phosphate salts, sodium salts, potassium salts,calcium salts, magnesium salts, and any other salt that can be found inblood or blood products, or that is known to be useful in dryingplatelets (e.g., anti-fibrinolytic loaded platelets), or any combinationof two or more of these.

In some embodiments, provided herein is composition comprising plateletssuch as lyophilized platelets or platelet derivatives (e.g.,thrombosomes), polysucrose and trehalose made by the process comprisingobtaining fresh platelets, optionally incubating the platelets in DMSO,isolating the platelets by centrifugation, resuspending the platelets inan incubating agent which comprises trehalose and ethanol therebyforming a first mixture, incubating the first mixture, mixingpolysucrose with the first mixture, thereby forming a second mixture,and lyophilizing the second mixture to form a freeze dried compositioncomprising platelets or platelet derivatives (e.g., thrombosomes),polysucrose and trehalose.

In some embodiments, provided herein is a method of making afreeze-dried platelet composition comprising platelets or plateletderivatives (e.g., thrombosomes), polysucrose and trehalose comprisingobtaining fresh platelets, optionally incubating the platelets in DMSO,isolating the platelets by centrifugation, resuspending the platelets ina incubating agent which comprises trehalose and ethanol thereby forminga first mixture, incubating the first mixture, mixing polysucrose withthe first mixture, thereby forming a second mixture, and lyophilizingthe second mixture to form a freeze-dried composition comprisingplatelets or platelet derivatives (e.g., thrombosomes), polysucrose andtrehalose.

In some embodiments, provided herein is a process for makingfreeze-dried platelets, the process comprising incubating isolatedplatelets in the presence of at least one saccharide under the followingconditions: a temperature of from 20° C. to 42° C. for about 10 minutesto about 180 minutes, adding to the platelets at least onecryoprotectant, and lyophilizing the platelets, wherein the processoptionally does not include isolating the platelets between theincubating and adding steps, and optionally wherein the process does notinclude exposing the platelets to a platelet activation inhibitor. Thecryoprotectant can be a polysugar (e.g., polysucrose). The process canfurther include heating the lyophilized platelets at a temperature of70° C. to 80° C. for 8 to 24 hours. The step of adding to the plateletsat least one cryoprotectant can further include exposing the plateletsto ethanol. The step of incubating isolated platelets in the presence ofat least one saccharide can include incubating in the presence of atleast one saccharide. The step of incubating isolated platelets in thepresence of at least one saccharide can include incubating in thepresence of at least one saccharide. The conditions for incubating caninclude incubating for about 100 minutes to about 150 minutes. Theconditions for incubating can include incubating for about 110 minutesto about 130 minutes. The conditions for incubating can includeincubating for about 120 minutes. The conditions for incubating caninclude incubating at 35° C. to 40° C. The conditions for incubating caninclude incubating at 37° C. The conditions for incubating can includeincubating at 35° C. to 40° C. for 110 minutes to 130 minutes. Theconditions for incubating can include incubating at 37° C. for 120minutes. The at least one saccharide can be trehalose, sucrose, or bothtrehalose and sucrose. The at least one saccharide can be trehalose. Theat least one saccharide can be sucrose.

In some embodiments, provided herein is a method of preparingfreeze-dried platelets, the method including providing platelets,suspending the platelets in a salt buffer that includes about 100 mMtrehalose and about 1% (v/v) ethanol to make a first composition,incubating the first composition at about 37° C. for about 2 hours,adding polysucrose (e.g., polysucrose 400) to a final concentration ofabout 6% (w/v) to make a second composition, lyophilizing the secondcomposition to make freeze-dried platelets, and heating the freeze-driedplatelets at 80° C. for 24 hours.

While the embodiments of the methods and compositions described hereinare amenable to various modifications and alternative forms, specificembodiments have been shown by way of example in the drawings and aredescribed in detail below. The intention, however, is not to limit themethods and compositions to the particular embodiments described. On thecontrary, the methods and compositions are intended to cover allmodifications, equivalents, and alternatives falling within the scope ofthe methods and compositions as defined by the appended claims.

EXAMPLES Example 1. Loading Platelets with ε-Aminocaproic Acid (EACA)

Protocol 1

The starting apheresis platelet material was pooled and acidified to apH 6.6-6.8 using 4 μL of 1M Acid Citrate Dextrose solution per 1 ml ofpooled platelet rich plasma. A count of platelets was obtained insolution using a Coulter AcT Diff hematology analyzer. The plateletswere isolated via centrifugation at 1500 g× for 20 minutes at roomtemperature with gentle acceleration and braking.

A dansyl-EACA/EACA solution was prepared as follows: stock of 1 mMdansyl-EACA and 1M EACA (1:1000 ratio), dissolved in loading buffer, andfrozen. The frozen dansyl-EACA/EACA solution was thawed at 37° C. for 20minutes.

The platelets were resuspended in loading buffer at a concentration of2,250,000 platelets/μL. The platelets (2,250,000 platelets/μl) werecombined with the EACA solution as follows: 9 ml platelets at 2,250,000platelets/μL and 1 ml dansyl-EACA/EACA stock as prepared above andincubated at 37° C. for 3 hours. 1 mL of dansyl-EACA/EACA stock and 30μL of 1 M dextrose were supplemented every hour on the hour.

The EACA-loaded platelets were cryopreserved by incubating the samplesin a freezer at −80° C. The cryopreserved EACA-loaded platelets werethawed at 37° C. water bath and used for downstream applicationsdescribed herein. A Tecan Infinite M200 Pro plate reader was used forquantification of anti-fibrinolytic loading. A TEG 5000 Hemostasisanalyzer was used to evaluate anti-fibrinolytic function of EACA fromEACA loaded platelets.

TABLE 1 Loading Buffer with EACA Concentration (mM, unless specifiedComponent otherwise) NaCl 750 KCl 48 HEPES 95 NaHCO₃ 120 Dextrose 3Trehalose 0.1 Ethanol 1.00% (v/v) Dansyl-EACA/ 0, 50, or 100 EACA(1:1000)

FIG. 1 is a graph showing dose-dependent EACA loading into platelets at50 mM and 100 mM over time measured at 1 hour, 2 hours, 3 hours, and 4hours. The pooled apheresis platelets were incubated with dansyl-EACA(λex/em: 325 nm, 570 nm) and unlabeled EACA at a molar ratio of 1:1000in loading buffer. Platelets were incubated for 1, 2, 3, or 4 hours at37° C. with low frequency agitation on a rocker. After isolation bycentrifugation and washing, the platelets were lysed by sonication andEACA per platelet was quantified using the Tecan Infinite® M200 PROplate reader. The results show dose-dependent and time-dependent EACAloading into platelets as measured by the concentration (mg/platelet) ofEACA per platelet in each sample.

Example 2. EACA-Loaded Platelet Functionality

FIG. 2 shows in vitro agonist stimulation of EACA release fromEACA-loaded platelets with known agonists: phorbol myristate acetate(PMA), collagen, or thrombin receptor activating peptide (TRAP)agonists. Platelets were loaded with EACA according to Protocol 1. EACAloaded platelets were incubated in either 1 μg/mL PMA, 10 μg/mLcollagen, or 10 μM TRAP in HMT buffer with 1 mM MgCl₂ for 10 minutes at37° C. to stimulate EACA release. After incubation, the platelets wereisolated as a pellet by centrifugation at 1470 g×10 minutes. Thereleased (supernatant) and intracellular (pellet) EACA concentrationswere quantified using the Tecan Infinite® M200 PRO plate reader.

FIG. 3 is a graph showing an EACA dose-response curve in pooled humanplatelet rich plasma to determine the effect of free EACA on lysis after30 minutes. Tissue plasminogen activator was used to induce fibrinolysisin vitro. 690 μL platelet rich plasma (platelets in George King plasma)was mixed with 10 μL of 65 μg/mL tPA and 10 μL of serially dilutedsamples of EACA in cell culture grade water. Each measurement was run induplicate using 340 μL of the EACA/tPA mixture pipetted into 20 μL of0.2 M CaCl₂). Runs were maintained for 30 minutes after MaximumAmplitude (MA) had been reached at 37° C. The percent lysis (LY30) showsthe extent of fibrinolysis 30 minutes after MA had been reached. A LY30of 100% corresponds to complete lysis of the blood clot while 0%corresponds to the absence of lysis. 140 μg/mL of free EACA was shown toeffectively inhibit fibrinolysis (LY30 of 3.5%).

FIG. 4 shows that EACA-loaded platelets can release EACA in vitro toprevent fibrinolysis. FIGS. 4A-E show thromboelastogram (TEG) graphs ofEACA loaded platelets with tissue plasminogen activator at varyingplatelet concentrations. Platelet concentrations tested were 125kcell/μL, 250 kcell/μL, 500 kcell/μL, 1000 kcell/μL, and 2000 kcell/μLof EACA-loaded platelets. Maximum amplitude was maintained over 30minutes with increasing concentration of drug loaded platelets. FIG. 4Fshows a dose-response curve of EACA-loaded platelets. The platelets werelysed and the concentration of EACA for each individual dose from A-E isplotted on the x-axis. A dose of EACA-loaded platelets equivalent to 2.5μg/mL prevents fibrinolysis (LY30 of 5.6%). 140 μg/mL free EACA inhibitfibrinolysis to a similar extent (LY30 of 3.5%). Therefore, EACA loadedinto platelets can inhibit tPA-induced coagulopathy at 56Δ lowerconcentrations than the free EACA.

FIG. 5 is a graph comparing the percent lysis of clots at 30 minutes forfree EACA in solution (FIG. 3) and EACA-loaded platelets (FIG. 4). FIG.5 shows, on a log scale, approximately equal percent lysis is observedusing platelet-loaded EACA at concentrations between 17 and 56-foldlower than for non-loaded EACA (free EACA). These results show thatplatelets are a viable vehicle for delivery of an anti-fibrinolytic suchas EACA at low concentrations relative to free EACA.

Example 3. Cryopreserved EACA-Loaded Platelets

FIG. 6 is a graph measuring the amount of EACA mg/platelet ofEACA-loaded platelets pre-cryopreservation and post-cryopreservation.Platelets were incubated in 100 mM dansyl-EACA (λex/em: 325 nm, 570 nm)and unlabeled EACA at a molar ratio of 1:1000 in loading buffer(Table 1) for 3 hours followed by centrifugation and resuspension inloading buffer containing sucrose and minimal DMSO for cryopreservation.The samples were cryopreserved by incubation of the samples in a −80° C.freezer. Drug load of pre- and post-cryopreservation were quantifiedusing a Tecan Infinite M200 Pro plate reader. The cryopreservedplatelets retained 91% of the original amount of EACA that was loadedper cell before preservation.

FIG. 7 shows that cryopreserved EACA-loaded platelets (EACA-loadedplatelets in the presence of loading buffer (Table 1) can release EACAin vitro to prevent fibrinolysis. FIGS. 7A-D show TEG graphs ofcryopreserved EACA loaded platelets plus tissue plasminogen activator atvarying platelet concentrations. Platelet concentrations ranged from 100kcell/μL, 250 kcell/μL, 500 kcell/μL, and 1000 kcell/μL. MA wasmaintained after 30 minutes with increasing concentration ofcryopreserved drug loaded platelets. FIG. 7E shows a dose-response curveof cryopreserved EACA loaded platelets. The platelets were lysed and theconcentration of EACA for each individual dose from A-E is plotted onthe x-axis. A dose of EACA-loaded platelets equivalent to 5 μg/mLprevents fibrinolysis (LY30 of 2.1%). 140 μg/mL free EACA inhibitfibrinolysis to a similar extent (LY30 of 3.5%). Therefore, EACA loadedinto platelets can inhibit tPA-induced coagulopathy at 28× lowerconcentrations than free EACA.

FIGS. 8A-C show graphs indicating the strength of clots as measured bymaximum amplitude (MA). FIG. 8A shows MA measured in the presence offree EACA. FIGS. 8B-C shows MA measured with EACA loaded platelets(5×10⁵ platelets/μl) in the presence of loading bufferpre-cryopreservation (8B) and post-cryopreservation (8C). MA increaseswith increasing number of EACA-loaded platelets in bothpre-cryopreservation and post-cryopreservation.

Example 4 Factor VIII and Factor IX Lengthen Activated PartialThromboplastin Time (aPTT)

Generally, a subject that takes an abnormally long time to form a bloodclot in a clinical aPTT assay can be a first diagnostic assay thatindicates a subject may have hemophilia or is lacking functional FactorVIII or Factor IX. FIG. 9 shows an aPTT analysis performed using plasmawith varying concentrations (0 μg/mL, 5 μg/mL, 10 μg/mL, & 20 μg/mL) ofantibodies that neutralize the activity of Factor VIII at roomtemperature or pre-incubated at 37° C. for two hours. It is known toperson skilled in the art that antibodies to Factor VIII are moreefficient at neutralization at higher temperatures typical in a subject.For example, it is known that sheep Factor VIII polyclonal antibodieswill cause an increase in clotting time at concentrations above 5 μg/mL.In contrast, higher concentrations of an antibody to Factor IX (40μg/mL) were required to cause an increase in clotting time. FIG. 10shows normal plasma samples incubated with varying concentrations (0μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, & 40 μg/mL) of antibodiesto Factor IX at room temperature or pre-incubated at 37° C. for twohours. An anti-Factor IX antibody concentration above 40 μg/mL increasedclotting time into the abnormal zone. Concentrations above 5 μg/mL ofanti-Factor VIII and above 40 μg/mL of anti-Factor IX were used to causeabnormal clotting times. The data demonstrate the proper concentrationof anti-Factor VIII and anti-Factor IX antibodies to use in subsequentassays necessary to recapitulate a clinically abnormal aPTT result.

Example 5 Antibodies to Factor VIII & Factor IX Reduce ThrombinProduction

A thrombin generation assay (TGA) stimulates and measures thrombinproduction. The TGA assay uses a coagulation stimulating agent combinedwith calcium to initiate coagulation and the resulting production ofthrombin. The presence of neutralizing antibodies to Factor VIII and/orIX inhibit full endogenous thrombin potential (ETP) from plasma bylimiting the intrinsic pathway of coagulation. As with the aPTT assay(Example 4) an antibody concentration to Factor VIII above 5 μg/mL andan antibody concentration to Factor IX above 50 μg/mL significantlyinhibit thrombin production. FIG. 11 shows ETP decreased withanti-Factor VIII antibodies in plasma. Normal plasma was incubated withvarious anti-Factor VIII antibodies and at 12.5 μg/mL completelyinhibited ETP. FIG. 12 shows a histogram of thrombin production inplasma samples incubated with Factor VIII antibodies at varyingconcentrations (0 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, &100 μg/mL). The data show an increase in time to peak thrombinconcentration and thrombin production was inhibited by the antibodies.Similarly, ETP was reduced with anti-Factor IX antibodies in plasma(FIG. 13). Normal plasma was treated with various anti-Factor IXantibodies which completely inhibited thrombin production at 100 μg/mLof antibody (FIG. 22). FIG. 14 shows a histogram of thrombin productionin plasma samples with anti-Factor IX antibodies. The data shown anincrease in time to peak thrombin concentration and thrombin productionwas inhibited by the antibodies.

Example 6 Thrombosomes Enhance the Rate of Thrombin Production in thePresence of Factor VIII Blocking Antibodies

Fresh platelet rich plasma was incubated with 10 μg/mL of Factor VIIIantibodies. The samples were run with or without 50,000 or 100,000thrombosomes per μL of sample. The length of time to peak thrombinproduction was increased by the Factor VIII antibodies, but the lengthof time was partially recovered with thrombosomes in subject 1 (FIGS.15-17) and subject 2 (FIGS. 18-20). Additionally, the concentration ofthrombin at peak production was decreased with Factor VIII antibodies,but it was partially recovered with the addition of thrombosomes.

FIG. 15 is a histogram showing thrombin generation in platelet richplasma from subject 1 was inhibited by Factor VIII antibodies andpartially recovered by thrombosomes. Freshly drawn platelet richplasma's ability to produce thrombin was inhibited with 10 μg/mL ofFactor VIII antibodies. The addition of thrombosomes partially recoveredplatelet rich plasma's ability to produce thrombin. FIG. 16 showspartial recovery of thrombin generation in platelet rich plasma fromsubject 1 in the presence of Factor VIII antibodies. Platelet richplasma treated with Factor VIII antibodies reduces peak thrombinproduction and also lengthens the time to reach peak thrombinproduction. The addition of thrombosomes recovered the length of time topeak thrombin production to normal and partially returned the peakthrombin concentrations. FIG. 17 shows a return to normal thrombingeneration potential of platelet rich plasma from subject 1 in thepresence of Factor VIII antibodies. Platelet rich plasma treated withFactor VIII antibodies reduces peak thrombin potential production. Theaddition of thrombosomes recovered the potential for thrombin productionto normal.

The experiment was replicated in a second subject (subject 2). FIG. 18shows a histogram of thrombin generation in platelet rich plasma fromsubject 2 was inhibited by Factor VIII antibodies and partiallyrecovered by the addition of thrombosomes. Freshly drawn platelet richplasma's ability to produce thrombin was inhibited with 10 μg/mL ofFactor VIII antibodies. The addition of thrombosomes partially recoveredplatelet rich plasma's ability to produce thrombin. FIG. 19 showspartial recovery of thrombin generation in platelet rich plasma fromsubject 2 in the presence of Factor VIII antibodies. Platelet richplasma treated with Factor VIII antibodies reduces peak thrombinproduction and lengthens the amount of time to peak thrombin production.The addition of thrombosomes recovered the amount of time to peakthrombin production to normal and partially recovered the peak thrombinlevels. FIG. 20 shows the return to normal thrombin generation potentialof platelet rich plasma from subject 2 in the presence of Factor VIIIantibodies. Platelet rich plasma treated with Factor VIII antibodiesreduces peak thrombin potential production and the addition ofthrombosomes recovered the potential for thrombin production to normal.

Example 7 Thrombosomes Enhance the Rate of Thrombin Production in thePresence of Factor IX Blocking Antibodies

Fresh platelet rich plasma was incubated with 100 μg/mL of Factor IXantibodies. The samples were run with or without 50,000, 100,000, or200,000 thrombosomes per μL of sample. The amount of time to peakthrombin production was increased by the Factor IX antibodies, however,the amount of time to peak thrombin production was partially recoveredwith thrombosomes in samples from subject 1 (FIGS. 21-23) and subject 2(FIGS. 24-26). Additionally, the concentration of thrombin at peak wasreduced with Factor IX antibodies, but that was partially recovered tonormal by the addition of thrombosomes

FIG. 21 shows a histogram of thrombin generation in platelet rich plasmafrom subject 1 was inhibited by Factor IX antibodies and partiallyrecovered by the addition of thrombosomes. Freshly drawn platelet richplasma's ability to produce thrombin was inhibited with 100 μg/mL ofFactor IX antibodies. The addition of thrombosomes partially recoveredthe platelet rich plasma's ability to produce thrombin. FIG. 22 showspartial recovery of thrombin generation in platelet rich plasma fromsubject 1 in the presence of Factor IX antibodies. Platelet rich plasmatreated with Factor IX antibodies reduces peak thrombin production andincreases the length of time to reach peak thrombin production. Theaddition of thrombosomes recovered the length of time to peak thrombinproduction to normal and partially recovered peak thrombinconcentration. FIG. 23 shows partial recovery to normal thrombingeneration potential of platelet rich plasma from subject 1 in thepresence of Factor IX antibodies. Platelet rich plasma treated withFactor IX antibodies reduces peak thrombin potential production,however, the addition of thrombosomes recovered the potential forthrombin production to normal.

The experiment was replicated in a second subject (subject 2). FIG. 24shows a histogram of thrombin generation in platelet rich plasma fromsubject 2 was inhibited by Factor IX antibodies and partially recoveredby the addition of thrombosomes. Freshly drawn platelet rich plasma'sability to produce thrombin was inhibited with 100 μg/mL of Factor IXantibodies. The addition of thrombosomes partially recovered plateletrich plasma's ability to produce thrombin. FIG. 25 shows partialrecovery of thrombin generation of platelet rich plasma from subject 2in the presence of Factor IX antibodies. Platelet rich plasma treatedwith Factor IX antibodies reduces peak thrombin production and lengthensthe amount of time to peak thrombin production. The addition ofthrombosomes recovered the time to peak thrombin production to normaland partially recovered peak thrombin concentration. FIG. 26 shows apartial return to normal thrombin generation potential of platelet richplasma from subject 2 in the presence of Factor IX antibodies. Plateletrich plasma treated with Factor IX antibodies reduces peak thrombinpotential production, however, the addition of thrombosomes recoveredthe potential for thrombin production to normal.

Example 8 Thrombosomes Reduce the Amount of Time to Start of ThrombinProduction in the Presence of Factor VIII Blocking Antibodies in a WholeBlood Sample

Whole blood was incubated with varying concentrations of antibodies toFactor VIII (0 μg/mL, 3 μg/mL, 6 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL,75 μg/mL, and 100 μg/mL) which increased the amount of time to thrombinproduction from 5 minutes to 9 minutes. The addition of thrombosomesrecovered the time to thrombin production to 5 minutes (FIG. 27).

Example 9 Clot Formation Restored in Thromboelastography (TEG) in Plasmawith Either Factor VIII Antibodies or Factor IX Antibodies withThrombosomes

A Thromboelastography assay monitors clot formation in small amounts ofplasma, platelet rich plasma, or whole blood. The instrument measuresthe resistance to movement in a pin submerged in the sample as the cupof sample moves. Clot formation parameters are measured including timeto clot initiation (r time), the rate of clot formation (K time), theangle of the clot, and the “MA” or “size of the clot.” FIG. 28 shows athromboelastography graph measuring the time to clot (r time), rate ofclot formation (k time), angle of the clot size, and MA size of theclot.

Normal fresh drawn plasma was treated with antibodies to Factor VIII(FIG. 29) or Factor IX (FIG. 30) at varying concentrations. Antibodiesto Factor VIII were added at 5 μg/mL, 10 μg/mL, and 20 μg/mL andincubated for 2 hours at 37° C. Antibodies to Factor IX were added at 50μg/mL, 100 μg/mL, and 150 μg/mL and incubated for 2 hours at 37° C. Eachsample was diluted one to one with PBS buffer (control) or 150 k/μL ofthrombosomes in PBS activated with Kaolin and placed into the TEG cupcontaining calcium chloride. The software recorded the test parameters.The addition of thrombosomes returned r-times in Factor VIII and FactorIX inhibited plasma to normal shown in FIGS. 28-30.

Thromboelastography Protocol:

The Thromboelastography was performed on the TEG5000 instrument fromHaemonetics(http://homepage.haemonetics.com/en/products/devices/surgical-and-diagnostic-devices/teg-5000).

Whole blood, plasma rich plasma or platelet poor plasma samples are usedas sample sources. Samples were pre-incubated with antibodies to eitherFactor VIII or Factor IX. The samples were mixed 1:1 with buffer (PBS orsimilar) or thrombosomes at 150 k/μL. 500 μL of sample+50 μL ofthrombosomes or buffer is added to Kaolin tubes with mixing. 20 μL ofcalcium chloride solution was added to each TEG cup on the instrument.340 μL of sample mixture from the Kaolin tube was added to a cup on theTEG instrument. Software was initiated and data collected.

Example 10—EACA-Loaded Fresh Platelets Restore Hemostasis in AcquiredHemophilia Types

FIG. 31 shows a model thromboelastography graph which measures theefficiency of blood coagulation and fibrinolysis. A thromboelastographygraph measures clotting time (“R time”), achievement of clot firmness(“K time”), clot development kinetics (“a” slope), and maximumamplitude.

FIG. 32 shows fresh platelets loaded with EACA restored hemostasis in aplatelet rich plasma model of acquired Hemophilia A (Factor VIIIdeficient). FIGS. 32A and B are positive and negative controls,respectively. Platelet rich plasma (500,000 cell/μl) was treated witheither unloaded platelets (FIG. 32A), unloaded platelets and an antibodyto coagulation Factor VIII (100 μg/mL) (FIG. 32B), unloaded plateletsand an antibody to coagulation Factor VIII (100 μg/mL) and free EACA(230 μg/mL) (FIG. 32C), and platelets loaded with EACA (5 μg/mL) and anantibody to coagulation Factor VIII (100 μg/mL) and free EACA (230μg/mL) (FIG. 32D). The experimental conditions are also shown in Table 2below.

The data show that treating platelet rich plasma in a model of acquiredHemophilia A with free EACA did not restore hemostasis (FIG. 32C). Incontrast, FIG. 32D shows restoration of hemostasis similar to thecontrol sample thromboelastography graph (FIG. 32A) when the plateletrich plasma model of acquired Hemophilia A was treated with plateletsloaded with 5 μg/mL of EACA (FIG. 32D). The numeric thromboelastographydata shown in FIGS. 32A-D are also summarized in Table 3 below. Thenumeric data also demonstrate that platelets loaded with EACA (5 μg/mL)restore hemostasis more effectively than free EACA alone (Table 3).

TABLE 2 Anti- Anti- Anti- Factor VIII Factor Factor Plasma Normal (100μg/mL) VIII VIII Cells Unloaded Unloaded Unloaded Loaded (500,000 μl)Platelets Platelets Platelets Platelets Free EACA N/A N/A 230 N/A(μg/mL) Loaded N/A N/A N/A 5 EACA (μg/mL)

TABLE 3 R time (sec) K time (sec) α (slope) Control 605 148 59.1 (+) αFactor VIII 1505 520 30.1 (++) = “+” Free EACA 1720 300 36.3 (*) = “+”Loaded EACA 1100 198 54.0

FIGS. 33A-E show fresh platelets loaded with EACA restored hemostasis ina platelet rich plasma model of acquired Hemophilia B (Factor IXdeficient). FIG. 33A shows a model thromboelastography graph in GeorgeKing Plasma (positive control) and FIG. 33C shows a thromboelastographygraph of plasma in a model of acquired Hemophilia B (100% lacking FactorIX). Both George King Plasma and acquired Hemophilia B model plateletrich plasma were treated with 140 μg/mL of free EACA (FIGS. 33B and 33D,respectively). The data show that free EACA on George King Plasma didnot affect hemostasis, whereas the free EACA partially restoredhemostasis in the acquired Hemophilia B plasma model (FIG. 33D). FIG.33E shows restoration of hemostasis similar to the positive control(FIG. 33A) when the platelet rich plasma model of acquired Hemophilia B(100% lacking Factor IX) was treated with EACA loaded platelets. Thedata in FIGS. 33A-E demonstrate that fresh platelets loaded with EACAare more effective at restoring hemostasis in a plasma model of acquiredHemophilia B than free EACA alone.

Example 11 EACA Loaded Thrombosomes in Acquired Hemophilia Models

A TGA was performed as described herein with an acquired Hemophilia Aplatelet rich plasma model. To perform the TGA assay a first solution of40 μL of thrombosomes in PBS, 40 μL of platelet rich plasma, and 20 μLof PPP-low were combined in triplicate wells. A PPP reagent stimulates aclotting cascade with low amounts of tissue factor and phospholipids.Additionally, a second solution of 40 μL of thrombosomes, 40 μL ofplatelet rich plasma, and 20 μL of a calibrator solution were combinedin triplicate wells. The platelet rich plasma in both the first solutionand the second solution was George King Plasma with either 7 μg/mL ofα-Factor VIII or 100 μg/mL of α-Factor IX. The addition of α-Factor VIIIor α-Factor IX to George King Plasma generates an acquired Hemophilia Aor an acquired Hemophilia B platelet rich plasma model, respectively.The first and the second solution were combined at incubated at 37° C.for 10 minutes with 20 μL of Fluo-substrate (FluCa). A Fluo-substratecontains a fluorogenic substrate solubilized in DMSO(https://www.thrombinoscope.com/method-products/products/). After the 10minute incubation the results were measured on a Thrombinoscope. TheEACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL,9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95%in either the acquired Hemophilia A platelet rich plasma model (FIG. 34)or the acquired Hemophilia B (data not shown) platelet rich plasmamodel.

Embodiments

Embodiment 1 is a method of treating acquired Hemophilia A in a subject,the method comprising: administering a therapeutically effective amountof anti-fibrinolytic loaded platelets to the subject in need thereof.

Embodiment 2 is a method of treating acquired Hemophilia B in a subject,the method comprising: administering a therapeutically effective amountof anti-fibrinolytic loaded platelets to the subject in need thereof.

Embodiment 3 is the method of any one of embodiments 1-2, wherein theconcentration of the therapeutically effective amount ofanti-fibrinolytic loaded into the platelets is from about 100 μM toabout 10 mM.

Embodiment 4 is a method of treating acquired Hemophilia A in a subject,the method comprising, administering a therapeutically effective amountof loaded thrombosomes to a subject in need thereof.

Embodiment 5 is a method of treating acquired Hemophilia B in a subject,the method comprising, administering a therapeutically effective amountof loaded thrombosomes to a subject in need thereof.

Embodiment 6 is the method of embodiment 4 or 5, wherein the loadedthrombosomes are loaded with an anti-fibrinolytic.

Embodiment 7 is the method of embodiment of 5 or 6, wherein theanti-fibrinolytic is selected from the group consisting ofε-aminocaproic acid, aprotinin, aminomethylbenzoic acid, tranexamicacid, and fibrinogen.

Embodiment 8 is the method of embodiment 7, wherein theanti-fibrinolytic is ε-aminocaproic acid.

Embodiment 9 is the method of embodiment 7 or 8, wherein ε-aminocaproicacid is present in a concentration from about 1 μM to about 100 mM.

Embodiment 10 is a method of treating acquired hemophilia A in asubject, the method comprising: administering a therapeuticallyeffective amount of unloaded thrombosomes to the subject in needthereof.

Embodiment 11 is a method of treating acquired hemophilia B in asubject, the method comprising: administering a therapeuticallyeffective amount of unloaded thrombosomes to the subject in needthereof.

Embodiment 12 is the method of embodiment 10 or 11, wherein theconcentration of the therapeutically effective amount of unloadedthrombosomes is from about 1×10² particles/kg to about 1×10¹³particles/kg.

Embodiment 13 is a method of treating a coagulopathy in a subject, themethod comprising administering to the subject in need thereof aneffective amount of a composition comprising platelets or plateletderivatives and an incubating agent comprising one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Embodiment 14 is a method of treating a coagulopathy in a subject, themethod comprising administering to the subject in need thereof aneffective amount of a composition prepared by a process comprisingincubating platelets with an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

Embodiment 15 is the method of embodiment 13 or 14, wherein thecomposition is administered following administration to the subject anantiplatelet agent or an anticoagulant, or a subject having acquiredhemophilia.

Embodiment 16 is a method of treating acquired hemophilia in a subjectin need thereof, comprising administering to the subjectanti-fibrinolytic loaded platelets, cryopreserved platelets, and/orfreeze-dried platelets according to Embodiment 1 or 2, wherein followingadministration peak thrombin (nM) is increased by at least 25%.

Embodiment 17 is a method of treating acquired hemophilia in a subjectin need thereof, comprising administering to the subjectanti-fibrinolytic loaded platelets, cryopreserved platelets, and/orfreeze-dried platelets according to Embodiment 1 or 2, wherein followingadministration time to peak thrombin (min) is decreased by at least 25%.

Embodiment 18 is a method of treating acquired hemophilia in a subjectin need thereof, comprising administering to the subjectanti-fibrinolytic loaded platelets, cryopreserved platelets, and/orfreeze-dried platelets according to Embodiment 1 or 2, wherein followingadministration the value of EPT in the plasma of the subject increasesby at least 25%.

1. A method of treating acquired Hemophilia in a subject, the methodcomprising: administering a therapeutically effective amount ofanti-fibrinolytic loaded platelets and/or a therapeutically effectiveamount of anti-fibrinolytic loaded thrombosomes to the subject in needthereof.
 2. (canceled)
 3. The method of claim 1, wherein the acquiredHemophilia is acquired Hemophilia A.
 4. The method of claim 1, whereinthe acquired Hemophilia is acquired Hemophilia B.
 5. The method of claim1, wherein the concentration of the therapeutically effective amount ofan anti-fibrinolytic loaded into the platelets is from about 100 μM toabout 10 mM.
 6. The method of claim 5, wherein the concentration of thetherapeutically effective amount of the anti-fibrinolytic loaded intothe platelets is from about 500 μM to about 5 mM.
 7. The method of claim6, wherein the concentration of the therapeutically effective amount ofthe anti-fibrinolytic loaded into the platelets is from about 1 mM toabout 3 mM.
 8. The method of claim 1, wherein the concentration of thetherapeutically effective amount of an anti-fibrinolytic loaded into theplatelets is at least 500 μM.
 9. A method of treating acquiredHemophilia in a subject, the method comprising: administering atherapeutically effective amount of anti-fibrinolytic loadedcryopreserved platelets to the subject in need thereof.
 10. The methodof claim 9, wherein the acquired Hemophilia is acquired Hemophilia A.11. The method of claim 9, wherein the acquired Hemophilia is acquiredHemophilia B.
 12. The method of claim 9, wherein the concentration ofthe therapeutically effective amount of anti-fibrinolytic loadedcryopreserved platelets is from about 1×10² particles/kg to about 1×10¹³particles/kg.
 13. The method of claim 12, wherein the concentration ofthe therapeutically effective amount of anti-fibrinolytic loadedcryopreserved platelets is from about 1×10⁴ particles/kg to about 1×10¹¹particles/kg.
 14. The method of claim 13, wherein the concentration ofthe therapeutically effective amount of anti-fibrinolytic loadedcryopreserved platelets is from about 1×10⁶ particles/kg to about 1×10⁹particles/kg.
 15. The method of claim 9, wherein the concentration ofthe therapeutically effective amount of anti-fibrinolytic loadedcryopreserved platelets or freeze dried platelets is at least 1×10⁴particles/kg.
 16. The method of claim 5, wherein the anti-fibrinolyticis selected from the group consisting of ε-aminocaproic acid, aprotinin,aminomethylbenzoic acid, tranexamic acid, and fibrinogen.
 17. The methodof claim 16, wherein the anti-fibrinolytic is ε-aminocaproic acid.
 18. Amethod of treating acquired Hemophilia A or acquired Hemophilia B,comprising administering a therapeutically effective amount ofanti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loadedplatelets are prepared by a process comprising contacting platelets withan anti-fibrinolytic and with a loading buffer including a salt, a base,a loading agent, and optionally at least one organic solvent, to formthe anti-fibrinolytic loaded platelets.
 19. A method to treat acquiredHemophilia A or acquired Hemophilia B comprising: A) pooling plateletsfrom a plurality of donors, B) contacting the platelets from step (A)with an anti-fibrinolytic and with a loading buffer including a salt, abase, a loading agent, and optionally at least one organic solvent, toform the anti-fibrinolytic loaded platelets and C) administering theanti-fibrinolytic loaded platelets to a subject in need thereof.
 20. Themethod of claim 1, wherein administering comprises administeringtopically, intravenously, intramuscularly, and combinations thereof. 21.The method of claim 1, wherein the method does not compriseadministering an anti-fibrinolytic.